iga  (Abcam)

 
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    Name:
    Goat anti Mouse C3 HRP Conjugated
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    AB208658
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    Structured Review

    Abcam iga
    The immune response of pigs after immunization with F4 fimbriae. The F4-specific IgG antibody responses ( A ), the F4-specific <t>IgA</t> antibody responses ( B ), number of F4-specifc total antibody secreting cells (ASCs) per 3.0 × 10 8 peripheral blood monomorphonuclear cells <t>(PBMCs)</t> ( C ), number of F4-specific IgA + ASCs per 3.0 × 10 8 PBMCs ( D ). Piglets were immunized with F4 fimbriae at day 0 and at 14 days post primary immunization (dppi). Data are presented as the mean ± SEM. The asterisk indicates a statistical significant difference ( p

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    Images

    1) Product Images from "Maternal immunity enhances systemic recall immune responses upon oral immunization of piglets with F4 fimbriae"

    Article Title: Maternal immunity enhances systemic recall immune responses upon oral immunization of piglets with F4 fimbriae

    Journal: Veterinary Research

    doi: 10.1186/s13567-015-0210-3

    The immune response of pigs after immunization with F4 fimbriae. The F4-specific IgG antibody responses ( A ), the F4-specific IgA antibody responses ( B ), number of F4-specifc total antibody secreting cells (ASCs) per 3.0 × 10 8 peripheral blood monomorphonuclear cells (PBMCs) ( C ), number of F4-specific IgA + ASCs per 3.0 × 10 8 PBMCs ( D ). Piglets were immunized with F4 fimbriae at day 0 and at 14 days post primary immunization (dppi). Data are presented as the mean ± SEM. The asterisk indicates a statistical significant difference ( p
    Figure Legend Snippet: The immune response of pigs after immunization with F4 fimbriae. The F4-specific IgG antibody responses ( A ), the F4-specific IgA antibody responses ( B ), number of F4-specifc total antibody secreting cells (ASCs) per 3.0 × 10 8 peripheral blood monomorphonuclear cells (PBMCs) ( C ), number of F4-specific IgA + ASCs per 3.0 × 10 8 PBMCs ( D ). Piglets were immunized with F4 fimbriae at day 0 and at 14 days post primary immunization (dppi). Data are presented as the mean ± SEM. The asterisk indicates a statistical significant difference ( p

    Techniques Used:

    The efficiency of using ELISpot on enriched IgA + B-cell fractions to enumerate F4-specific IgA + antibody secreting cells (ASCs). A In one well, only one F4-specific IgA + ASC was detected from 5 × 10 5 peripheral blood monomorphonuclear cells (PBMCs) (left) while 18 F4-specific spots were detected from 5 × 10 5 enriched IgA + B-cells (right) at 21 days post primary immunization (dppi) of an oral immunized pig having maternal antibodies; each blue spot is counted as one F4-specific IgA + ASC. B From eight wells, less than seven F4-specific IgA + ASCs were detected from 4.0 × 10 6 PBMCs whereas more than 28 F4-specific IgA + ASCs were detected from 4.0 × 10 6 enriched IgA + B-cells at 21 dppi of the three immunized groups. The asterisk indicates a statistical significant difference ( p
    Figure Legend Snippet: The efficiency of using ELISpot on enriched IgA + B-cell fractions to enumerate F4-specific IgA + antibody secreting cells (ASCs). A In one well, only one F4-specific IgA + ASC was detected from 5 × 10 5 peripheral blood monomorphonuclear cells (PBMCs) (left) while 18 F4-specific spots were detected from 5 × 10 5 enriched IgA + B-cells (right) at 21 days post primary immunization (dppi) of an oral immunized pig having maternal antibodies; each blue spot is counted as one F4-specific IgA + ASC. B From eight wells, less than seven F4-specific IgA + ASCs were detected from 4.0 × 10 6 PBMCs whereas more than 28 F4-specific IgA + ASCs were detected from 4.0 × 10 6 enriched IgA + B-cells at 21 dppi of the three immunized groups. The asterisk indicates a statistical significant difference ( p

    Techniques Used: Enzyme-linked Immunospot

    2) Product Images from "Anti-Leptospira immunoglobulin profiling in mice reveals strain specific IgG and persistent IgM responses associated with virulence and renal colonization"

    Article Title: Anti-Leptospira immunoglobulin profiling in mice reveals strain specific IgG and persistent IgM responses associated with virulence and renal colonization

    Journal: bioRxiv

    doi: 10.1101/2020.11.26.399667

    Homologous challenge boosts the specific anti- Leptospira humoral immunity including IgG1 and IgG3 responses A) Profiles of anti- Leptospira IgM and IgG antibodies after serovar specific challenge in mice immunized with L. interrogans Manilae or Copenhageni or Icterohaemorrhagiae strains. Specific IgM (upper panels) and specific IgA (lower panels) produced after Manilae bioluminescent derivative MFLum1 challenge in mice initially immunized with Manilae L495 or M895 (left panels), or after homologous challenge in mice initially immunized with Copenhageni Fiocruz LV2756 (central panels), or after Icterohaemorrhagiae Verdun challenge in mice immunized with virulent Verdun or avirulent (AV) (right panels). B) Profiles of four specific anti- Leptospira IgG subclasses elicited after serovar specific challenge in mice immunized with L. interrogans Manilae or Copenhageni or Icterohaemorrhagiae strains. IgG1, IgG3, IgG2b, IgG2c antibodies produced after Manilae bioluminescent derivative MFLum1 challenge in mice initially immunized with Manilae L495 or M895 strains (left panels), or after homologous challenge in mice initially immunized with Fiocruz LV2756 (central panels), or Icterohaemorrhagiae Verdun challenge in mice immunized with virulent Verdun or avirulent (right panels). Control corresponds to mice initially injected with PBS and challenged with the respective virulent strain specific to each experimental condition and PBS/PBS (in Verdun groups) corresponds to mice injected with PBS instead pre-infection and challenge as negative control. Each figure represents the profiles for IgM and IgA isotypes and IgG subclasses obtained from serum tested in pool (for a same experimental group). Antibody responses were assessed up to D55 p.C. (Manilae groups), D28 p.C. (Copenhageni groups) and D78 p.C. (Icterohaemorrhagiae groups) with n=5 female mice/group with exception of control Manilae group (n=4; one mouse died after challenge).
    Figure Legend Snippet: Homologous challenge boosts the specific anti- Leptospira humoral immunity including IgG1 and IgG3 responses A) Profiles of anti- Leptospira IgM and IgG antibodies after serovar specific challenge in mice immunized with L. interrogans Manilae or Copenhageni or Icterohaemorrhagiae strains. Specific IgM (upper panels) and specific IgA (lower panels) produced after Manilae bioluminescent derivative MFLum1 challenge in mice initially immunized with Manilae L495 or M895 (left panels), or after homologous challenge in mice initially immunized with Copenhageni Fiocruz LV2756 (central panels), or after Icterohaemorrhagiae Verdun challenge in mice immunized with virulent Verdun or avirulent (AV) (right panels). B) Profiles of four specific anti- Leptospira IgG subclasses elicited after serovar specific challenge in mice immunized with L. interrogans Manilae or Copenhageni or Icterohaemorrhagiae strains. IgG1, IgG3, IgG2b, IgG2c antibodies produced after Manilae bioluminescent derivative MFLum1 challenge in mice initially immunized with Manilae L495 or M895 strains (left panels), or after homologous challenge in mice initially immunized with Fiocruz LV2756 (central panels), or Icterohaemorrhagiae Verdun challenge in mice immunized with virulent Verdun or avirulent (right panels). Control corresponds to mice initially injected with PBS and challenged with the respective virulent strain specific to each experimental condition and PBS/PBS (in Verdun groups) corresponds to mice injected with PBS instead pre-infection and challenge as negative control. Each figure represents the profiles for IgM and IgA isotypes and IgG subclasses obtained from serum tested in pool (for a same experimental group). Antibody responses were assessed up to D55 p.C. (Manilae groups), D28 p.C. (Copenhageni groups) and D78 p.C. (Icterohaemorrhagiae groups) with n=5 female mice/group with exception of control Manilae group (n=4; one mouse died after challenge).

    Techniques Used: Mouse Assay, Produced, Injection, Infection, Negative Control

    Sustained and persistent specific antibodies, including IgM that correlate with presence of leptospires in kidneys. anti- Leptospira immunoglobulins generated after experimental infection of C57BL/6J mice with representative strains of four distinct serovars (Manilae, Copenhageni, Icterohaemorrhagiae and Patoc) of leptospires. A) Kinetics and isotyping determination over a six months period (28 days for Patoc) after infection. Mice were intraperitoneally inoculated with 2×10 7 of virulent Manilae L495 strain (dark blue circle) or avirulent Manilae M895 mutant (light blue square), or virulent Copenhageni Fiocruz LV2756 strain (dark green circle) or virulent Icterohaemorrhagiae Verdun strain (dark pink circle) or avirulent (AV) strain (light pink square) or saprophytic Patoc Patoc 1 strain (orange circle), or PBS as negative control. B) Profiles of 3 specific different isotypes (IgM, IgA, IgG) produced over the first month after experimental infection with different doses of virulent strains (Icterohaemorrhagiae Verdun or Manilae L495 strains) or inactivated heat killed (HK) Manilae L495 strain. Mice were intraperitoneally inoculated with 2×10 8 (empty pink circle) or with 2×10 7 (dark pink circle) of virulent Icterohaemorrhagiae Verdun, or with 1×10 5 of virulent Manilae L495 strain (dashed blue line) or 2×10 7 of heat-inactivated L495 (grey circle) or 2×10 7 (dark blue circle) of virulent Manilae L495, or with PBS as negative control. Each figure represents the profiles for total Igs or each specific isotypes obtained from serum tested in pool, for a same experimental group, at D0, D3 post-infection (p.i.) or individually (when error bars), each dot being a determined time-point p.i. Absence of visible error bars post D8 p.i., notably for Icterohaemorrhagiae and Patoc patterns performed with individual samples, is due to the scale. Specific Ig responses were assessed up to day 180 p.i. with female mice (n=5/group) or day 28 p.i. with male mice (n=5/group). C) Leptospiral loads in kidneys of mice 28 days after intraperitoneal infection with representative strains of three distinct pathogenic (Manilae, Copenhageni, Icterohaemorrhagiae) and one saprophytic (Patoc) serovars. Mice were inoculated with 2×10 7 of virulent Manilae L495 or bioluminescent Manilae L495 MFLum1 derivative strains or avirulent Manilae M895 mutant, or virulent Copenhageni Fiocruz LV2756 strain or virulent Icterohaemorrhagiae Verdun strain (2 doses) or avirulent (AV) strain, or saprophytic Patoc 1 strain (serovar Patoc), or PBS as negative control. After euthanasia at D28 p.i., kidneys were collected and DNA purification performed before qPCR amplification. Each dot corresponds to one sample, n=5/group.
    Figure Legend Snippet: Sustained and persistent specific antibodies, including IgM that correlate with presence of leptospires in kidneys. anti- Leptospira immunoglobulins generated after experimental infection of C57BL/6J mice with representative strains of four distinct serovars (Manilae, Copenhageni, Icterohaemorrhagiae and Patoc) of leptospires. A) Kinetics and isotyping determination over a six months period (28 days for Patoc) after infection. Mice were intraperitoneally inoculated with 2×10 7 of virulent Manilae L495 strain (dark blue circle) or avirulent Manilae M895 mutant (light blue square), or virulent Copenhageni Fiocruz LV2756 strain (dark green circle) or virulent Icterohaemorrhagiae Verdun strain (dark pink circle) or avirulent (AV) strain (light pink square) or saprophytic Patoc Patoc 1 strain (orange circle), or PBS as negative control. B) Profiles of 3 specific different isotypes (IgM, IgA, IgG) produced over the first month after experimental infection with different doses of virulent strains (Icterohaemorrhagiae Verdun or Manilae L495 strains) or inactivated heat killed (HK) Manilae L495 strain. Mice were intraperitoneally inoculated with 2×10 8 (empty pink circle) or with 2×10 7 (dark pink circle) of virulent Icterohaemorrhagiae Verdun, or with 1×10 5 of virulent Manilae L495 strain (dashed blue line) or 2×10 7 of heat-inactivated L495 (grey circle) or 2×10 7 (dark blue circle) of virulent Manilae L495, or with PBS as negative control. Each figure represents the profiles for total Igs or each specific isotypes obtained from serum tested in pool, for a same experimental group, at D0, D3 post-infection (p.i.) or individually (when error bars), each dot being a determined time-point p.i. Absence of visible error bars post D8 p.i., notably for Icterohaemorrhagiae and Patoc patterns performed with individual samples, is due to the scale. Specific Ig responses were assessed up to day 180 p.i. with female mice (n=5/group) or day 28 p.i. with male mice (n=5/group). C) Leptospiral loads in kidneys of mice 28 days after intraperitoneal infection with representative strains of three distinct pathogenic (Manilae, Copenhageni, Icterohaemorrhagiae) and one saprophytic (Patoc) serovars. Mice were inoculated with 2×10 7 of virulent Manilae L495 or bioluminescent Manilae L495 MFLum1 derivative strains or avirulent Manilae M895 mutant, or virulent Copenhageni Fiocruz LV2756 strain or virulent Icterohaemorrhagiae Verdun strain (2 doses) or avirulent (AV) strain, or saprophytic Patoc 1 strain (serovar Patoc), or PBS as negative control. After euthanasia at D28 p.i., kidneys were collected and DNA purification performed before qPCR amplification. Each dot corresponds to one sample, n=5/group.

    Techniques Used: Generated, Infection, Mouse Assay, Mutagenesis, Negative Control, Produced, DNA Purification, Real-time Polymerase Chain Reaction, Amplification

    3) Product Images from "Sulfur dioxide exposure reduces the quantity of CD19+ cells and causes nasal epithelial injury in rats"

    Article Title: Sulfur dioxide exposure reduces the quantity of CD19+ cells and causes nasal epithelial injury in rats

    Journal: Journal of Occupational Medicine and Toxicology (London, England)

    doi: 10.1186/s12995-018-0205-x

    The representative figure showed that SO 2 inhalation did not affect IgG levels in the serum and nasal septum. a : IgG level in the serum; b : IgG level in the nasal septum; c : IgE level in the serum; d : IgE level in the nasal septum; e : IgA level in the serum; f : IgA level in the nasal septum
    Figure Legend Snippet: The representative figure showed that SO 2 inhalation did not affect IgG levels in the serum and nasal septum. a : IgG level in the serum; b : IgG level in the nasal septum; c : IgE level in the serum; d : IgE level in the nasal septum; e : IgA level in the serum; f : IgA level in the nasal septum

    Techniques Used:

    4) Product Images from "Curdlan sulfate–O-linked quaternized chitosan nanoparticles: potential adjuvants to improve the immunogenicity of exogenous antigens via intranasal vaccination"

    Article Title: Curdlan sulfate–O-linked quaternized chitosan nanoparticles: potential adjuvants to improve the immunogenicity of exogenous antigens via intranasal vaccination

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S158536

    Humoral immunoresponse of immunized mice. Notes: ( A ) Relative expression of IgG in serum; ( B ) relative expression of IgA in serum; ( C ) expression of subtype antibodies in serum. * P
    Figure Legend Snippet: Humoral immunoresponse of immunized mice. Notes: ( A ) Relative expression of IgG in serum; ( B ) relative expression of IgA in serum; ( C ) expression of subtype antibodies in serum. * P

    Techniques Used: Mouse Assay, Expressing

    5) Product Images from "Elucidation of the Effects of a Current X-SCID Therapy on Intestinal Lymphoid Organogenesis Using an In Vivo Animal Model"

    Article Title: Elucidation of the Effects of a Current X-SCID Therapy on Intestinal Lymphoid Organogenesis Using an In Vivo Animal Model

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    doi: 10.1016/j.jcmgh.2020.01.011

    The effect of BMT for X-SCID pigs on the development of the lymphoid tissue structure and intestinal immune and microbial environments. ( A ) IgG was detected in the serum of both age-matched WT pigs (n = 3) and BMT-treated X-SCID pigs (n = 3). ( B ) Histological analyses showed that only a few ILFs, which are composed of abnormally accumulated CD3 + T cells and CD20 + B cells, developed in the BMT-treated X-SCID pigs. ( B ) One trial (BMT3), using a GFP-expressing transgenic pig as the donor of BMT, showed that the ILFs were composed of both donor and recipient cells because some (not all) cells in the ILFs were recognized by the anti-GFP. ( C ) In addition, abundant levels of Igs, especially IgA, were found in the feces of WT pigs, whereas only 1 of 3 BMT-treated X-SCID pigs had IgA in the feces. Immunohistochemical analyses showed that few class-switched IgA + cells were present in the ILFs. ( D ) However, detectable levels of IgA + cells were scattered in the villi of all 3 BMT-treated X-SCID pigs. ( E ) A metagenomics analysis demonstrated that the intestinal microflora was not identical between BMT-treated X-SCID pigs and age-matched WT pigs. Scale bars = 100 μm. HE, hematoxylin and eosin.
    Figure Legend Snippet: The effect of BMT for X-SCID pigs on the development of the lymphoid tissue structure and intestinal immune and microbial environments. ( A ) IgG was detected in the serum of both age-matched WT pigs (n = 3) and BMT-treated X-SCID pigs (n = 3). ( B ) Histological analyses showed that only a few ILFs, which are composed of abnormally accumulated CD3 + T cells and CD20 + B cells, developed in the BMT-treated X-SCID pigs. ( B ) One trial (BMT3), using a GFP-expressing transgenic pig as the donor of BMT, showed that the ILFs were composed of both donor and recipient cells because some (not all) cells in the ILFs were recognized by the anti-GFP. ( C ) In addition, abundant levels of Igs, especially IgA, were found in the feces of WT pigs, whereas only 1 of 3 BMT-treated X-SCID pigs had IgA in the feces. Immunohistochemical analyses showed that few class-switched IgA + cells were present in the ILFs. ( D ) However, detectable levels of IgA + cells were scattered in the villi of all 3 BMT-treated X-SCID pigs. ( E ) A metagenomics analysis demonstrated that the intestinal microflora was not identical between BMT-treated X-SCID pigs and age-matched WT pigs. Scale bars = 100 μm. HE, hematoxylin and eosin.

    Techniques Used: Expressing, Transgenic Assay, Immunohistochemistry

    The effect of BMT to patients with X-SCID on the development of the lymphoid tissue structure and intestinal immune and microbial environments. Serum samples were collected from BMT-treated patients with X-SCID (n = 5) and used to assess the amounts of Igs (ie, IgM, IgG, and IgA). ( A ) Although 2 of 5 patients (Nos. 1 and 4) were receiving continuous IVIG, all patients had serum Igs, especially IgG. ( B ) Magnetic resonance imaging revealed that the 2 patients receiving continuous IVIG did not possess an obvious thymic structure. ( B ) One patient (No. 2), 29 years of age, developed the thymus, but the structure was atrophied and replaced mostly with adipose tissue. Arrowheads show the thymus. Fecal samples were collected from BMT-treated patients with X-SCID (n = 5) and used to assess the amounts of Igs (ie, IgM, IgG, and IgA) and the development of intestinal microflora. ( C ) No Igs were detected in fecal samples from the 2 patients (Nos. 1 and 4) receiving IVIG, and only small amounts of IgA were detected in feces from 2 other patients (Nos. 3 and 5). ( C ) By contrast, sufficient IgA was detected in feces from 1 patient (No. 2). ( D ) A metagenomics analysis demonstrated that only the patient who produced a high amount of IgA in feces developed a diverse intestinal microflora.
    Figure Legend Snippet: The effect of BMT to patients with X-SCID on the development of the lymphoid tissue structure and intestinal immune and microbial environments. Serum samples were collected from BMT-treated patients with X-SCID (n = 5) and used to assess the amounts of Igs (ie, IgM, IgG, and IgA). ( A ) Although 2 of 5 patients (Nos. 1 and 4) were receiving continuous IVIG, all patients had serum Igs, especially IgG. ( B ) Magnetic resonance imaging revealed that the 2 patients receiving continuous IVIG did not possess an obvious thymic structure. ( B ) One patient (No. 2), 29 years of age, developed the thymus, but the structure was atrophied and replaced mostly with adipose tissue. Arrowheads show the thymus. Fecal samples were collected from BMT-treated patients with X-SCID (n = 5) and used to assess the amounts of Igs (ie, IgM, IgG, and IgA) and the development of intestinal microflora. ( C ) No Igs were detected in fecal samples from the 2 patients (Nos. 1 and 4) receiving IVIG, and only small amounts of IgA were detected in feces from 2 other patients (Nos. 3 and 5). ( C ) By contrast, sufficient IgA was detected in feces from 1 patient (No. 2). ( D ) A metagenomics analysis demonstrated that only the patient who produced a high amount of IgA in feces developed a diverse intestinal microflora.

    Techniques Used: Magnetic Resonance Imaging, Produced

    Lack of antibody production in the ileum of X-SCID pigs. ( A ) Tissue sections of ileum harvested from WT (n = 5) and X-SCID (n = 5) pigs were stained with either anti-IgA, anti-IgG, or anti-IgM. In the PPs of WT pigs, IgA + cells were abundant, and there were some IgM + and a few IgG + cells above the follicular area, in which B cells expressing surface IgM were present. In the lamina propria of intestinal villi, a large number of IgA + cells, some IgM + cells, and a few IgG + cells were found. However, such Ig + cells were rarely detected in the ileum of X-SCID pigs. ( B ) The amounts of Igs (ie, IgA, IgG, IgM) in feces from WT (n = 8) and X-SCID (n = 7) pigs were measured by ELISA. WT pigs produced a large amount of IgA, followed by IgM and IgG in their feces, whereas X-SCID pigs had mostly undetectable levels of Igs in feces. Asterisks show the follicular area. Scale bars = 100 μm. * P
    Figure Legend Snippet: Lack of antibody production in the ileum of X-SCID pigs. ( A ) Tissue sections of ileum harvested from WT (n = 5) and X-SCID (n = 5) pigs were stained with either anti-IgA, anti-IgG, or anti-IgM. In the PPs of WT pigs, IgA + cells were abundant, and there were some IgM + and a few IgG + cells above the follicular area, in which B cells expressing surface IgM were present. In the lamina propria of intestinal villi, a large number of IgA + cells, some IgM + cells, and a few IgG + cells were found. However, such Ig + cells were rarely detected in the ileum of X-SCID pigs. ( B ) The amounts of Igs (ie, IgA, IgG, IgM) in feces from WT (n = 8) and X-SCID (n = 7) pigs were measured by ELISA. WT pigs produced a large amount of IgA, followed by IgM and IgG in their feces, whereas X-SCID pigs had mostly undetectable levels of Igs in feces. Asterisks show the follicular area. Scale bars = 100 μm. * P

    Techniques Used: Staining, Expressing, Enzyme-linked Immunosorbent Assay, Produced

    6) Product Images from "IgA transcytosis and antigen recognition govern ovarian cancer immunity"

    Article Title: IgA transcytosis and antigen recognition govern ovarian cancer immunity

    Journal: Nature

    doi: 10.1038/s41586-020-03144-0

    IgA internalization by HGSOC cells is associated with stronger T cell responses, and is dependent on pIgR–Fc interactions. a , Representative ( n = 273) IgA and IgG staining in tumours with a high or low density of CD4 + and CD8 + T cells. Scale bar, 200 μm or 100 μm, as indicated. CD4 + and CD8 + T cell accumulation (≥median) is associated with the density of IgA-coated tumour (PCK + ) cells (IgA: CD4
    Figure Legend Snippet: IgA internalization by HGSOC cells is associated with stronger T cell responses, and is dependent on pIgR–Fc interactions. a , Representative ( n = 273) IgA and IgG staining in tumours with a high or low density of CD4 + and CD8 + T cells. Scale bar, 200 μm or 100 μm, as indicated. CD4 + and CD8 + T cell accumulation (≥median) is associated with the density of IgA-coated tumour (PCK + ) cells (IgA: CD4

    Techniques Used: Staining

    Multiplex immunohistochemistry staining optimization for CD3, CD4, CD8, CD19, CD20, CD138, IgA, IgG, pIgR and PCK in tonsil tissues. a , b , Multiplex staining of tonsil tissue sections ( n = 5) with anti-human CD3, CD4, CD8, CD19, CD20 and CD138 antibodies or respective isotype controls ( a ), and IgA, IgG, pIgR and PCK antibodies or respective isotype controls ( b ). Scale bar, 100 μm.
    Figure Legend Snippet: Multiplex immunohistochemistry staining optimization for CD3, CD4, CD8, CD19, CD20, CD138, IgA, IgG, pIgR and PCK in tonsil tissues. a , b , Multiplex staining of tonsil tissue sections ( n = 5) with anti-human CD3, CD4, CD8, CD19, CD20 and CD138 antibodies or respective isotype controls ( a ), and IgA, IgG, pIgR and PCK antibodies or respective isotype controls ( b ). Scale bar, 100 μm.

    Techniques Used: Multiplex Assay, Immunohistochemistry, Staining

    TSPAN7- and BDNF-specific antibodies produced at tumour beds delay the progression of established tumours. a , RAG1-deficient mice inoculated subcutaneously with 10 7 OVCAR3 cells received 100 μg per 20g body weight of irrelevant (i)IgA or irrelevant (i)IgG peritumourally at days 7, 11, 15, 19 and 23 after tumour inoculation. Tumour growth curves (left), tumour weight (centre) and representative differences in tumour volume (right) are shown. Growth curve statistics: iIgG versus iIgA, P = 0.0130, paired two-tailed t -test. Tumour weight statistics, iIgG versus iIgA, P = 0.043, unpaired two-tailed t -test. Data are mean ± s.e.m. * P ≤ 0.05. b , Tumour growth curves (left), as well as tumour volume (right) and weight (centre) in OVCAR3-tumour-bearing RAG1-deficient mice receiving irrelevant IgG antibodies or vehicle (PBS). Growth curve statistics: iIgG versus PBS, P = 0.1840, paired two-tailed t -test. Tumour weight statistics: iIgG versus PBS, P = 0.8275, (unpaired two-tailed t -test). Data are mean ± s.e.m. NS, not significant. c , Tumour growth curves (left), as well as tumour volume (right) and weight (centre) in OVCAR3-tumour-bearing RAG1-deficient mice receiving full-length or pepsinized (Fc-removed) irrelevant IgG or irrelevant IgA antibodies. Curves and tumour weights were pooled from 2 independent experiments (10 mice per group in total). Growth curve statistics: iIgA versus F(ab′) 2 –iIgA, P = 0.0030; iIgA versus iIgG, P = 0.0578; iIgG versus F(ab′) 2 –iIgG, P = 0.0547, paired two-tailed t -test. Tumour weight statistics: iIgA versus F(ab′) 2 –iIgA, P
    Figure Legend Snippet: TSPAN7- and BDNF-specific antibodies produced at tumour beds delay the progression of established tumours. a , RAG1-deficient mice inoculated subcutaneously with 10 7 OVCAR3 cells received 100 μg per 20g body weight of irrelevant (i)IgA or irrelevant (i)IgG peritumourally at days 7, 11, 15, 19 and 23 after tumour inoculation. Tumour growth curves (left), tumour weight (centre) and representative differences in tumour volume (right) are shown. Growth curve statistics: iIgG versus iIgA, P = 0.0130, paired two-tailed t -test. Tumour weight statistics, iIgG versus iIgA, P = 0.043, unpaired two-tailed t -test. Data are mean ± s.e.m. * P ≤ 0.05. b , Tumour growth curves (left), as well as tumour volume (right) and weight (centre) in OVCAR3-tumour-bearing RAG1-deficient mice receiving irrelevant IgG antibodies or vehicle (PBS). Growth curve statistics: iIgG versus PBS, P = 0.1840, paired two-tailed t -test. Tumour weight statistics: iIgG versus PBS, P = 0.8275, (unpaired two-tailed t -test). Data are mean ± s.e.m. NS, not significant. c , Tumour growth curves (left), as well as tumour volume (right) and weight (centre) in OVCAR3-tumour-bearing RAG1-deficient mice receiving full-length or pepsinized (Fc-removed) irrelevant IgG or irrelevant IgA antibodies. Curves and tumour weights were pooled from 2 independent experiments (10 mice per group in total). Growth curve statistics: iIgA versus F(ab′) 2 –iIgA, P = 0.0030; iIgA versus iIgG, P = 0.0578; iIgG versus F(ab′) 2 –iIgG, P = 0.0547, paired two-tailed t -test. Tumour weight statistics: iIgA versus F(ab′) 2 –iIgA, P

    Techniques Used: Produced, Mouse Assay, Two Tailed Test

    IgA–pIgR colocalization is associated with protective immunity in human ovarian cancer. a , Left, percentage of FACS cell counts of IgA + , IgG + or IgM + cells among Ig + B cells or plasmablasts or plasma cells, normalized to 10,000 viable CD45 + cells. B cells, CD45 + CD3 − CD19 + CD20 + cells; plasmablasts, CD45 + CD3 − CD19 + CD20 − CD38 high cells; plasma cells, CD45 + CD3 − CD19 + CD20 − CD138 + and CD45 + CD3 − CD19 − CD20 − CD138 + cells. Each dot represents one tumour ( n = 29). Details of box plots can be found in Methods. P values were obtained by a two-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparisons. Supplementary Table 1 provides further details on statistics. Right, bar graphs representing the percentage of each isotype produced by plasma cells (top) or B cells (bottom) in the same tumours, normalized to 10,000 viable CD45 + cells. IC, intracellular. b , IgA-coated CD45 − EpCAM + tumour epithelial cells (mean ± s.e.m., n = 10) in dissociated HGSOC. c , Expression of pIgR protein in independent HGSOC ( n = 27); tumour-free Fallopian tube ( n = 3), ovary ( n = 5) and omental ( n = 4) samples; ovarian tumour cell lines; and K562 leukaemia cells and THP1 monocyte cells (negative controls). Positive control, recombinant human pIgR. Western blots were repeated twice. NSCLC, non-small-cell lung cancer. d , Histograms showing FACS analysis of pIgR, in ovarian surface epithelial (OSE), K562, THP1, wild-type or PIGR -ablated (pIgR CRISPR ) OVCAR3 cells. e , Left, representative ( n = 273) combined staining of IgA, pIgR, IgG, PCK and DAPI. Instances with IgA–pIgR colocalization are indicated with arrows. Scale bar, 50 μm (top left), 20 μm (all other panels). Top right, representative ( n = 137, IgA–pIgR colocalization ≥ median) dot plot showing IgA–pIgR colocalized signal among DAPI + PCK + cells. Bottom right, scattered graph showing number of IgA–pIgR colocalized cells (averaged from duplicated cores) per mm 2 of PCK + (mean ± s.e.m., n = 273). f , Increased numbers of cells with IgA–pIgR colocalization per PCK + tumour islet area (averaged from duplicated cores) are associated with improved outcome (threshold, median; P = 0.0116, H. Lee Moffett Cancer Centre cohort (MCC) (right); P = 0.0002, New England Case–Control study cohort (NECC) (left)). g , Density of IgA-coated cells (averaged from duplicated cores) in tumour islets (cells per mm 2 PCK + area) is associated with improved outcome ( P = 0.0110 for MCC (right) and P = 0.0054 for NECC (left) cohorts). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, two-sided log-rank (Mantel–Cox) test. Source data
    Figure Legend Snippet: IgA–pIgR colocalization is associated with protective immunity in human ovarian cancer. a , Left, percentage of FACS cell counts of IgA + , IgG + or IgM + cells among Ig + B cells or plasmablasts or plasma cells, normalized to 10,000 viable CD45 + cells. B cells, CD45 + CD3 − CD19 + CD20 + cells; plasmablasts, CD45 + CD3 − CD19 + CD20 − CD38 high cells; plasma cells, CD45 + CD3 − CD19 + CD20 − CD138 + and CD45 + CD3 − CD19 − CD20 − CD138 + cells. Each dot represents one tumour ( n = 29). Details of box plots can be found in Methods. P values were obtained by a two-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparisons. Supplementary Table 1 provides further details on statistics. Right, bar graphs representing the percentage of each isotype produced by plasma cells (top) or B cells (bottom) in the same tumours, normalized to 10,000 viable CD45 + cells. IC, intracellular. b , IgA-coated CD45 − EpCAM + tumour epithelial cells (mean ± s.e.m., n = 10) in dissociated HGSOC. c , Expression of pIgR protein in independent HGSOC ( n = 27); tumour-free Fallopian tube ( n = 3), ovary ( n = 5) and omental ( n = 4) samples; ovarian tumour cell lines; and K562 leukaemia cells and THP1 monocyte cells (negative controls). Positive control, recombinant human pIgR. Western blots were repeated twice. NSCLC, non-small-cell lung cancer. d , Histograms showing FACS analysis of pIgR, in ovarian surface epithelial (OSE), K562, THP1, wild-type or PIGR -ablated (pIgR CRISPR ) OVCAR3 cells. e , Left, representative ( n = 273) combined staining of IgA, pIgR, IgG, PCK and DAPI. Instances with IgA–pIgR colocalization are indicated with arrows. Scale bar, 50 μm (top left), 20 μm (all other panels). Top right, representative ( n = 137, IgA–pIgR colocalization ≥ median) dot plot showing IgA–pIgR colocalized signal among DAPI + PCK + cells. Bottom right, scattered graph showing number of IgA–pIgR colocalized cells (averaged from duplicated cores) per mm 2 of PCK + (mean ± s.e.m., n = 273). f , Increased numbers of cells with IgA–pIgR colocalization per PCK + tumour islet area (averaged from duplicated cores) are associated with improved outcome (threshold, median; P = 0.0116, H. Lee Moffett Cancer Centre cohort (MCC) (right); P = 0.0002, New England Case–Control study cohort (NECC) (left)). g , Density of IgA-coated cells (averaged from duplicated cores) in tumour islets (cells per mm 2 PCK + area) is associated with improved outcome ( P = 0.0110 for MCC (right) and P = 0.0054 for NECC (left) cohorts). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, two-sided log-rank (Mantel–Cox) test. Source data

    Techniques Used: FACS, Produced, Expressing, Positive Control, Recombinant, Western Blot, CRISPR, Staining

    Transcytosis of IgA through pIgR + ovarian cancer cells impairs tumour growth and augments cytotoxic killing mediated by T cells. a , Left, images of APC-labelled IgA binding and internalization in pIgR + OVCAR3 cells (repeated three times). Scale bar, 50 μm (main panels), 10 μm (magnified regions). Right, comparison of antibody internalization signal (mean ± s.e.m.) in different treatment conditions and at different temporal points. Each dot represents quantification from one cell. *** P ≤ 0.001, unpaired two-tailed t -test. Supplementary Table 2 provides details of statistics. b , OVCAR3 cells were incubated with control IgA or IgG for 8 h in the presence of wortmannin, brefeldin A (BFA) or vehicle, and supernatants were subjected to liquid chromatography with tandem mass spectrometry (LC–MS/MS). Heat map of all peptides of the extracellular domain of pIgR ( n = 3); scale represents log 2 -transformed intensities of pIgR peptide fragments detected in LC–MS/MS. c , Left, co-immunoprecipitates of supernatants from IgA-treated pIgR + or PIGR -ablated OVCAR3 cells (with and without brefeldin A or wortmannin) blotted for the secretory component of pIgR and IgA (input control). Right, LC–MS/MS analysis of the co-immunoprecipitates showing intensities (log 2 -transformed) of the secretory component of pIgR and IgA ( n = 2). WT, wild type; CR, PIGR -ablated. d , Pre-ranked gene-set enrichment analysis (GSEA), showing the top upregulated gene sets in OVCAR3 cells treated with irrelevant IgA compared to IgG or untreated cells ( n = 3), Kolmogorov–Smirnov test. GO, Gene Ontology. e , Progressive increase in DUSP5 and concomitant reduction in phospho-ERK1 and phospho-ERK2 (pERK1/2) after IgA treatment (left) of OVCAR3 cells, but not IgG treatment (right). Experiments were repeated three times. tERK1/2, total ERK1 and total ERK2. f , Left, dose-dependent cytotoxic killing of NY-ESO-1-transduced OVCAR3 cells ( n = 3) by NY-ESO-1–TCR-transduced T cells is augmented by co-incubation with IgA, compared to IgG or PBS. Right, IgA treatment also augmented the anti-tumour activity of FSH-targeted chimeric receptor T cells. Mean ± s.e.m. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, ordinary one-way ANOVA. Supplementary Table 2 provides details of statistics. g , Cytotoxic killing of primary CD45 − EpCAM + tumour cells ( n = 3) by autologous tumour-infiltrating T cells (1:1 ratio) is augmented by co-incubation with autologous ( P
    Figure Legend Snippet: Transcytosis of IgA through pIgR + ovarian cancer cells impairs tumour growth and augments cytotoxic killing mediated by T cells. a , Left, images of APC-labelled IgA binding and internalization in pIgR + OVCAR3 cells (repeated three times). Scale bar, 50 μm (main panels), 10 μm (magnified regions). Right, comparison of antibody internalization signal (mean ± s.e.m.) in different treatment conditions and at different temporal points. Each dot represents quantification from one cell. *** P ≤ 0.001, unpaired two-tailed t -test. Supplementary Table 2 provides details of statistics. b , OVCAR3 cells were incubated with control IgA or IgG for 8 h in the presence of wortmannin, brefeldin A (BFA) or vehicle, and supernatants were subjected to liquid chromatography with tandem mass spectrometry (LC–MS/MS). Heat map of all peptides of the extracellular domain of pIgR ( n = 3); scale represents log 2 -transformed intensities of pIgR peptide fragments detected in LC–MS/MS. c , Left, co-immunoprecipitates of supernatants from IgA-treated pIgR + or PIGR -ablated OVCAR3 cells (with and without brefeldin A or wortmannin) blotted for the secretory component of pIgR and IgA (input control). Right, LC–MS/MS analysis of the co-immunoprecipitates showing intensities (log 2 -transformed) of the secretory component of pIgR and IgA ( n = 2). WT, wild type; CR, PIGR -ablated. d , Pre-ranked gene-set enrichment analysis (GSEA), showing the top upregulated gene sets in OVCAR3 cells treated with irrelevant IgA compared to IgG or untreated cells ( n = 3), Kolmogorov–Smirnov test. GO, Gene Ontology. e , Progressive increase in DUSP5 and concomitant reduction in phospho-ERK1 and phospho-ERK2 (pERK1/2) after IgA treatment (left) of OVCAR3 cells, but not IgG treatment (right). Experiments were repeated three times. tERK1/2, total ERK1 and total ERK2. f , Left, dose-dependent cytotoxic killing of NY-ESO-1-transduced OVCAR3 cells ( n = 3) by NY-ESO-1–TCR-transduced T cells is augmented by co-incubation with IgA, compared to IgG or PBS. Right, IgA treatment also augmented the anti-tumour activity of FSH-targeted chimeric receptor T cells. Mean ± s.e.m. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, ordinary one-way ANOVA. Supplementary Table 2 provides details of statistics. g , Cytotoxic killing of primary CD45 − EpCAM + tumour cells ( n = 3) by autologous tumour-infiltrating T cells (1:1 ratio) is augmented by co-incubation with autologous ( P

    Techniques Used: Binding Assay, Two Tailed Test, Incubation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Transformation Assay, Activity Assay

    Multiplex immunohistochemistry staining optimization for CD3, CD4, CD8, CD19, CD20, CD138, IgA, IgG, pIgR and PCK in glioblastoma tissues, and for pIgR in kidney tissues. a , b , Multiplex staining of glioblastoma tissue sections ( n = 5) with anti-human CD3, CD4, CD8, CD19, CD20 and CD138 antibodies ( a ), and IgA, IgG, pIgR and PCK antibodies ( b ). Scale bar, 100 μm. c , Multiplex staining of healthy kidney tissue sections ( n = 4) with anti-human pIgR antibody and respective isotype control. Scale bar, 100 μm.
    Figure Legend Snippet: Multiplex immunohistochemistry staining optimization for CD3, CD4, CD8, CD19, CD20, CD138, IgA, IgG, pIgR and PCK in glioblastoma tissues, and for pIgR in kidney tissues. a , b , Multiplex staining of glioblastoma tissue sections ( n = 5) with anti-human CD3, CD4, CD8, CD19, CD20 and CD138 antibodies ( a ), and IgA, IgG, pIgR and PCK antibodies ( b ). Scale bar, 100 μm. c , Multiplex staining of healthy kidney tissue sections ( n = 4) with anti-human pIgR antibody and respective isotype control. Scale bar, 100 μm.

    Techniques Used: Multiplex Assay, Immunohistochemistry, Staining

    IgA transcytosis through HGSOC cells has substantial anti-tumour effects and sensitizes tumour cells to cytolytic killing by T cells. a , Immunoblots showing pIgR co-immunoprecipitation with IgA, using nondenaturing lysates from two HGSOCs, CD45 + and CD45 − cells isolated from human ovarian cancer ascites, ascitic fluid and OVCAR3 cells (negative control). Inputs were immunoblotted using 1% of the amount of lysate used for the co-immunoprecipitation. The experiments were repeated three times. b , OVCAR3 cells were incubated with 0.5 μg ml −1 of control IgA or IgG for 8 h in serum-free medium in the presence of wortmannin (1 μM), brefeldin A (1 μg ml −1 ) or vehicle, and supernatants were subjected to mass spectrometry. The amino acids 62–77 fragment of pIgR was found only after incubation with IgA (repeated three times). c–e , OVCAR4 ( c ), OVCAR5 ( d ) and primary HGSOC ( e ) tumour cells were incubated with 0.5 μg ml −1 of irrelevant IgA or IgG for 8 h in serum-free medium in the presence of wortmannin (1 μM), brefeldin A (1 μg ml −1 ) or vehicle, and supernatants were then subjected to mass spectrometry (left). Right, heat map of all peptides of the extracellular domain of pIgR ( n = 3). BFA, brefeldin A. WM, wortmannin. f , g , GSEA enrichment plots ( h ) and heat maps ( g ) using normalized gene expression from RNA-seq analysis from OVCAR3 cells with irrelevant IgA (0.5 μg ml −1 ), IgG (0.5 μg ml −1 ) or no treatment for 24 h ( n = 3 experiments). h , No change in the protein levels of DUSP5, total ERK1/2 or phospho-ERK1/2 after vehicle (PBS) treatment of pIgR + (WT) OVACR3 cells (left) or after IgA treatment of PIGR -ablated OVACR3 cells (right), incubated up to 8 h. The experiments were repeated three times. i , Dose-dependent cytotoxic killing of OVCAR3 cells by FSH-targeted chimeric receptor T cells is augmented by co-incubation with 0.5 μg ml −1 of irrelevant IgA, anti-TSPAN7–IgA or anti-BDNF–IgA compared to IgG, pepsinized irrelevant IgA or PBS. n = 3 per group. ** P ≤ 0.01, unpaired two-tailed t -test. j , Cytotoxic killing of autologous CD45 − EpCAM + tumour cells (with corresponding decrease of annexin V − propidium iodide (PI) − viable cells) by autologous T cells (added at 1:1 ratio) is augmented by co-incubation with 0.5 μg ml −1 of autologous IgA or irrelevant IgA but not with autologous IgG, pepsinized autologous or irrelevant IgA, as compared to uncoated cells ( n = 3). Annexin V + : irrelevant IgA versus pepsinized irrelevant IgA, P
    Figure Legend Snippet: IgA transcytosis through HGSOC cells has substantial anti-tumour effects and sensitizes tumour cells to cytolytic killing by T cells. a , Immunoblots showing pIgR co-immunoprecipitation with IgA, using nondenaturing lysates from two HGSOCs, CD45 + and CD45 − cells isolated from human ovarian cancer ascites, ascitic fluid and OVCAR3 cells (negative control). Inputs were immunoblotted using 1% of the amount of lysate used for the co-immunoprecipitation. The experiments were repeated three times. b , OVCAR3 cells were incubated with 0.5 μg ml −1 of control IgA or IgG for 8 h in serum-free medium in the presence of wortmannin (1 μM), brefeldin A (1 μg ml −1 ) or vehicle, and supernatants were subjected to mass spectrometry. The amino acids 62–77 fragment of pIgR was found only after incubation with IgA (repeated three times). c–e , OVCAR4 ( c ), OVCAR5 ( d ) and primary HGSOC ( e ) tumour cells were incubated with 0.5 μg ml −1 of irrelevant IgA or IgG for 8 h in serum-free medium in the presence of wortmannin (1 μM), brefeldin A (1 μg ml −1 ) or vehicle, and supernatants were then subjected to mass spectrometry (left). Right, heat map of all peptides of the extracellular domain of pIgR ( n = 3). BFA, brefeldin A. WM, wortmannin. f , g , GSEA enrichment plots ( h ) and heat maps ( g ) using normalized gene expression from RNA-seq analysis from OVCAR3 cells with irrelevant IgA (0.5 μg ml −1 ), IgG (0.5 μg ml −1 ) or no treatment for 24 h ( n = 3 experiments). h , No change in the protein levels of DUSP5, total ERK1/2 or phospho-ERK1/2 after vehicle (PBS) treatment of pIgR + (WT) OVACR3 cells (left) or after IgA treatment of PIGR -ablated OVACR3 cells (right), incubated up to 8 h. The experiments were repeated three times. i , Dose-dependent cytotoxic killing of OVCAR3 cells by FSH-targeted chimeric receptor T cells is augmented by co-incubation with 0.5 μg ml −1 of irrelevant IgA, anti-TSPAN7–IgA or anti-BDNF–IgA compared to IgG, pepsinized irrelevant IgA or PBS. n = 3 per group. ** P ≤ 0.01, unpaired two-tailed t -test. j , Cytotoxic killing of autologous CD45 − EpCAM + tumour cells (with corresponding decrease of annexin V − propidium iodide (PI) − viable cells) by autologous T cells (added at 1:1 ratio) is augmented by co-incubation with 0.5 μg ml −1 of autologous IgA or irrelevant IgA but not with autologous IgG, pepsinized autologous or irrelevant IgA, as compared to uncoated cells ( n = 3). Annexin V + : irrelevant IgA versus pepsinized irrelevant IgA, P

    Techniques Used: Western Blot, Immunoprecipitation, Isolation, Negative Control, Incubation, Mass Spectrometry, Expressing, RNA Sequencing Assay, Two Tailed Test

    7) Product Images from "A booster dose enhances immunogenicity of the COVID-19 vaccine candidate ChAdOx1 nCoV-19 in aged mice"

    Article Title: A booster dose enhances immunogenicity of the COVID-19 vaccine candidate ChAdOx1 nCoV-19 in aged mice

    Journal: bioRxiv

    doi: 10.1101/2020.10.27.357426

    ChAdOx1 nCoV-19 induces a plasma cell and germinal centre B cell response a. tSNE/FlowSOM analyses of CD19 + B cells from 3-month-old (3mo) mice seven days after immunization with ChAdOx1 nCoV-19 or PBS. Heatmaps of the manually gated B cell populations indicated at seven, 14 and 21 days after immunisation in the aortic lymph node ( b ) and spleen ( c ). Here the frequency of each cell subset in each ChAdOx1 nCoV-19 immunised mouse has been expressed as the log2 fold change over the average frequency in PBS immunised mice (n=5). Bar charts showing the total number of plasma cells ( d ), CD69 + IgD + B cells ( e ), proliferating non-germinal centre B cells ( f ) and germinal centre B cells ( g ) at the indicated time points after immunization. Number of T follicular helper ( h ) and T follicular regulatory cells ( i ) at the indicated time points postimmunisation. Serum anti-spike IgM, IgG, and IgA ( j ) antibodies seven, 14 and 21 days after immunization. k . Pie charts indicating the mean abundance of each IgG antibody subclass in the serum at the indicated time points after immunisation. In d-j bar height in corresponds to the mean and each circle represents one biological replicate. P-values are calculated using a student’s t-test with Holm-Sidak multiple testing correction, for ELISA data analyses were done on log transformed values.
    Figure Legend Snippet: ChAdOx1 nCoV-19 induces a plasma cell and germinal centre B cell response a. tSNE/FlowSOM analyses of CD19 + B cells from 3-month-old (3mo) mice seven days after immunization with ChAdOx1 nCoV-19 or PBS. Heatmaps of the manually gated B cell populations indicated at seven, 14 and 21 days after immunisation in the aortic lymph node ( b ) and spleen ( c ). Here the frequency of each cell subset in each ChAdOx1 nCoV-19 immunised mouse has been expressed as the log2 fold change over the average frequency in PBS immunised mice (n=5). Bar charts showing the total number of plasma cells ( d ), CD69 + IgD + B cells ( e ), proliferating non-germinal centre B cells ( f ) and germinal centre B cells ( g ) at the indicated time points after immunization. Number of T follicular helper ( h ) and T follicular regulatory cells ( i ) at the indicated time points postimmunisation. Serum anti-spike IgM, IgG, and IgA ( j ) antibodies seven, 14 and 21 days after immunization. k . Pie charts indicating the mean abundance of each IgG antibody subclass in the serum at the indicated time points after immunisation. In d-j bar height in corresponds to the mean and each circle represents one biological replicate. P-values are calculated using a student’s t-test with Holm-Sidak multiple testing correction, for ELISA data analyses were done on log transformed values.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Transformation Assay

    8) Product Images from "Recirculating Intestinal IgA-Producing Cells Regulate Neuroinflammation via IL-10"

    Article Title: Recirculating Intestinal IgA-Producing Cells Regulate Neuroinflammation via IL-10

    Journal: Cell

    doi: 10.1016/j.cell.2018.11.035

    IgA-Binding of Bacteria in the Gut Is Reduced in MS Patients during an Acute Relapse (A) Study overview: fresh-frozen fecal samples of 33 patients with clinically Isolated syndrome (CIS) or relapslng-remlttlng multiple sclerosis (RRMS) (n = 22 during disease remission, n = 11 during an acute relapse) and 32 healthy controls were collected. Fecal bacteria were isolated and IgA-binding of autologous gut bacteria was quantified by bacterial flow cytometry (BUGFlow) and ELISA. (B) BUGFlow: bacteria were identified based on forward and side scatter and the IgA-positive population was defined based on an isotype control. Shown is an example of staining of fecal bacteria from an RRMS patient with an isotype control (left) as well as with anti-IgA during remission (middle) and during relapse (right). (C) Percentage of fecal-bound IgA by BUG Flow: 1 × 10 6 fecal bacteria were stained with anti-IgA and % of IgA binding was assessed by flow cytometry. Values shown are replicates from 2 experiments. (D) Quantification of fecal-bound IgA by ELISA: 1 × 10 6 fecal bacteria were assessed for IgA binding using a commercially available quantitative IgA ELISA assay. Values shown are mean values of duplicate measurements. *p
    Figure Legend Snippet: IgA-Binding of Bacteria in the Gut Is Reduced in MS Patients during an Acute Relapse (A) Study overview: fresh-frozen fecal samples of 33 patients with clinically Isolated syndrome (CIS) or relapslng-remlttlng multiple sclerosis (RRMS) (n = 22 during disease remission, n = 11 during an acute relapse) and 32 healthy controls were collected. Fecal bacteria were isolated and IgA-binding of autologous gut bacteria was quantified by bacterial flow cytometry (BUGFlow) and ELISA. (B) BUGFlow: bacteria were identified based on forward and side scatter and the IgA-positive population was defined based on an isotype control. Shown is an example of staining of fecal bacteria from an RRMS patient with an isotype control (left) as well as with anti-IgA during remission (middle) and during relapse (right). (C) Percentage of fecal-bound IgA by BUG Flow: 1 × 10 6 fecal bacteria were stained with anti-IgA and % of IgA binding was assessed by flow cytometry. Values shown are replicates from 2 experiments. (D) Quantification of fecal-bound IgA by ELISA: 1 × 10 6 fecal bacteria were assessed for IgA binding using a commercially available quantitative IgA ELISA assay. Values shown are mean values of duplicate measurements. *p

    Techniques Used: Binding Assay, Mass Spectrometry, Isolation, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Staining

    9) Product Images from "In planta expression of nanobody-based designer chicken antibodies targeting Campylobacter"

    Article Title: In planta expression of nanobody-based designer chicken antibodies targeting Campylobacter

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0204222

    Screening for the expression of chimeric antibodies in A . thaliana seeds using SDS-PAGE (A, C) and western blot (B, D). The expression of chimeric antibodies in seeds was confirmed for extracts positive in ELISA. Western blots were developed with a mouse anti-histidine tag monoclonal antibody and a goat anti-mouse antibody conjugated to HRP. As negative controls, extract of wild-type seeds and the V1G chimeric antibody lacking the His-tag [ 23 ] were used. A His-tagged nanobody was used as a positive control, resulting in a protein band with a molecular weight of approximately 15 kDa. (A) IgA chimeric antibodies with V1, Nb23 and Nb5, (C) IgY chimeric antibodies with V1, Nb23 and Nb5.
    Figure Legend Snippet: Screening for the expression of chimeric antibodies in A . thaliana seeds using SDS-PAGE (A, C) and western blot (B, D). The expression of chimeric antibodies in seeds was confirmed for extracts positive in ELISA. Western blots were developed with a mouse anti-histidine tag monoclonal antibody and a goat anti-mouse antibody conjugated to HRP. As negative controls, extract of wild-type seeds and the V1G chimeric antibody lacking the His-tag [ 23 ] were used. A His-tagged nanobody was used as a positive control, resulting in a protein band with a molecular weight of approximately 15 kDa. (A) IgA chimeric antibodies with V1, Nb23 and Nb5, (C) IgY chimeric antibodies with V1, Nb23 and Nb5.

    Techniques Used: Expressing, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, Positive Control, Molecular Weight

    T-DNA construct used for the expression of the chimeric antibodies. (LB) left border, (3’OCS) octopine synthase terminator, ( npt II) neomycin phosphotransferase II gene, (P nos ) nopaline synthase promoter, (Pphas) β-phaseolin promoter, ( att B1 att B2) attachment sites for Gateway recombination, (2S2) signal peptide of the 2S2 seed storage protein, (Nb-Fc) Fusion of anti- Campylobacter nanobody to chicken IgA or IgY, (His) histidine-tag, (KDEL) endoplasmic retention peptide, (3’ arc5-1 ) arcelin terminator and (RB) right border.
    Figure Legend Snippet: T-DNA construct used for the expression of the chimeric antibodies. (LB) left border, (3’OCS) octopine synthase terminator, ( npt II) neomycin phosphotransferase II gene, (P nos ) nopaline synthase promoter, (Pphas) β-phaseolin promoter, ( att B1 att B2) attachment sites for Gateway recombination, (2S2) signal peptide of the 2S2 seed storage protein, (Nb-Fc) Fusion of anti- Campylobacter nanobody to chicken IgA or IgY, (His) histidine-tag, (KDEL) endoplasmic retention peptide, (3’ arc5-1 ) arcelin terminator and (RB) right border.

    Techniques Used: Construct, Expressing

    Visualisation of the binding of chimeric anti-MOMP antibodies in seed extract with Campylobacter isolates. (A, B, C, J, K, L) C . jejuni strain KC40, (D, E, F, M, N, O) C . jejuni strain Cam12/0156 and (G, H, I, P, Q, R) C . coli strain K43/5. Binding with the different Campylobacter isolates is shown with seed extract containing (A, D, G) Nb5-IgA, (B, E, H) Nb23-IgA, (C, F, I) V1-IgA, (J, M, P) Nb5-IgY, (K, N, Q) Nb23-IgY and (L, O, R) V1-IgY. As a negative control, the nanobody V1 against F4-fimbriated E . coli was used. Bright field microscopy was used for the visualisation of the corresponding bacterial cells.
    Figure Legend Snippet: Visualisation of the binding of chimeric anti-MOMP antibodies in seed extract with Campylobacter isolates. (A, B, C, J, K, L) C . jejuni strain KC40, (D, E, F, M, N, O) C . jejuni strain Cam12/0156 and (G, H, I, P, Q, R) C . coli strain K43/5. Binding with the different Campylobacter isolates is shown with seed extract containing (A, D, G) Nb5-IgA, (B, E, H) Nb23-IgA, (C, F, I) V1-IgA, (J, M, P) Nb5-IgY, (K, N, Q) Nb23-IgY and (L, O, R) V1-IgY. As a negative control, the nanobody V1 against F4-fimbriated E . coli was used. Bright field microscopy was used for the visualisation of the corresponding bacterial cells.

    Techniques Used: Binding Assay, Negative Control, Microscopy

    Expression of chimeric antibodies in transgenic A . thaliana seeds. Extracts of T2 seeds were coated and analysed by ELISA. The presence of chimeric antibodies in the extracts was tested, using anti-chicken IgA or IgY conjugated to HRP. As a negative control, extract of wild-type A . thaliana seeds was used. The results of seed extracts from A . thaliana plants transformed with Nb5-IgA, Nb23-IgA, V1-IgA, Nb5-IgY, Nb23-IgY and V1-IgY are shown.
    Figure Legend Snippet: Expression of chimeric antibodies in transgenic A . thaliana seeds. Extracts of T2 seeds were coated and analysed by ELISA. The presence of chimeric antibodies in the extracts was tested, using anti-chicken IgA or IgY conjugated to HRP. As a negative control, extract of wild-type A . thaliana seeds was used. The results of seed extracts from A . thaliana plants transformed with Nb5-IgA, Nb23-IgA, V1-IgA, Nb5-IgY, Nb23-IgY and V1-IgY are shown.

    Techniques Used: Expressing, Transgenic Assay, Enzyme-linked Immunosorbent Assay, Negative Control, Transformation Assay

    Binding of chimeric antibodies to C . jejuni KC40 bacteria and purified MOMP. KC40 bacteria and MOMP (1 μ g/ml) were coated in an ELISA plate. Subsequently, the interaction of twofold serial dilutions of the seed extracts was assessed. Therefore, anti-IgA and anti-IgY antibodies, conjugated to HRP, were used for the detection of chimeric antibodies, bound to the bacteria or to the purified native MOMP antigen. As a negative control, the binding of extracts of wild-type A . thaliana seeds with the bacteria and MOMP was measured. (A) Nb5-IgA, (B) Nb23-IgA, (C) Nb5-IgY and (D) Nb23-IgY. The error bars correspond to the standard deviations.
    Figure Legend Snippet: Binding of chimeric antibodies to C . jejuni KC40 bacteria and purified MOMP. KC40 bacteria and MOMP (1 μ g/ml) were coated in an ELISA plate. Subsequently, the interaction of twofold serial dilutions of the seed extracts was assessed. Therefore, anti-IgA and anti-IgY antibodies, conjugated to HRP, were used for the detection of chimeric antibodies, bound to the bacteria or to the purified native MOMP antigen. As a negative control, the binding of extracts of wild-type A . thaliana seeds with the bacteria and MOMP was measured. (A) Nb5-IgA, (B) Nb23-IgA, (C) Nb5-IgY and (D) Nb23-IgY. The error bars correspond to the standard deviations.

    Techniques Used: Binding Assay, Purification, Enzyme-linked Immunosorbent Assay, Negative Control

    Transient expression of chimeric antibodies carrying Nb5 in leaves of N . benthamiana . Protein extracts were analysed using (A) SDS-PAGE stained with Coomassie blue dye and (B) western blot developed with mouse anti-histidine tag monoclonal antibody. Extracts of N . benthamiana leaves transformed with Nb5-IgA and Nb5-IgY fusion constructs were tested. Leaves infiltrated with A . tumefaciens harbouring the vector pEAQ -HT were used as negative control.
    Figure Legend Snippet: Transient expression of chimeric antibodies carrying Nb5 in leaves of N . benthamiana . Protein extracts were analysed using (A) SDS-PAGE stained with Coomassie blue dye and (B) western blot developed with mouse anti-histidine tag monoclonal antibody. Extracts of N . benthamiana leaves transformed with Nb5-IgA and Nb5-IgY fusion constructs were tested. Leaves infiltrated with A . tumefaciens harbouring the vector pEAQ -HT were used as negative control.

    Techniques Used: Expressing, SDS Page, Staining, Western Blot, Transformation Assay, Construct, Plasmid Preparation, Negative Control

    Interaction of chimeric antibodies in seed extract from homozygous plants with their antigen. SDS-PAGE and a western blot were performed on purified MOMP under non-denaturing conditions. (A) SDS-PAGE with purified native MOMP stained with Coomassie blue dye. The results of the western blot confirm the interactions of (B) Nb23-IgA14D and (C) Nb23-IgY12C, with MOMP. (D) Wild-type extract was used as a negative control. The western blot was developed with anti-IgA or anti-IgY antibodies conjugated to HRP.
    Figure Legend Snippet: Interaction of chimeric antibodies in seed extract from homozygous plants with their antigen. SDS-PAGE and a western blot were performed on purified MOMP under non-denaturing conditions. (A) SDS-PAGE with purified native MOMP stained with Coomassie blue dye. The results of the western blot confirm the interactions of (B) Nb23-IgA14D and (C) Nb23-IgY12C, with MOMP. (D) Wild-type extract was used as a negative control. The western blot was developed with anti-IgA or anti-IgY antibodies conjugated to HRP.

    Techniques Used: SDS Page, Western Blot, Purification, Staining, Negative Control

    10) Product Images from "Fatty acid-induced CD36 expression via O-GlcNAcylation drives gastric cancer metastasis"

    Article Title: Fatty acid-induced CD36 expression via O-GlcNAcylation drives gastric cancer metastasis

    Journal: Theranostics

    doi: 10.7150/thno.34024

    Upregulation of CD36 in GC cell promotes cell metastasis. (A) Flow cytometry analysis showing the expression of CD36 in SGC 7901-NM cells transfected with a lentiviral vector encoding CD36 or negative control vector. K isotype IgG was used as a negative control. (B)3D spheroid BME cell invasion assay of SGC 7901-NM cells transfected with lentiviral vector encoding CD36 (LV-CD36) or with negative control vector (LV-NC). Photographs of all the spheroids in each well every 24 h for 7 days using a 4× objective. Quantitative analysis of the surface area of all spheroids. Normalized areas for all the spheroids are presented relative to the area on the first day. All of the areas were calculated three times using ImageJ, and the values represent the means ± SD. (C) The indicated cells were injected into nude mice (n = 6 for each group) via the tail vein along with weekly intraperitoneal injections of 20 μg of the anti-CD36 neutralizing monoclonal antibody JC63.1 or 20 μg of the corresponding IgA. Animals were sacrificed at 8 weeks after the injections. Photos of representative lung tissue samples in each group are shown. (D) Left: The histogram shows the proportion of mice with lung metastasis in each group. “Met” is short for “metastasis,” and “Met-free” indicates “metastasis-free.” Right: Mann Whitney test was used to evaluate the number of metastatic nodes in the lungs of mice from each group.
    Figure Legend Snippet: Upregulation of CD36 in GC cell promotes cell metastasis. (A) Flow cytometry analysis showing the expression of CD36 in SGC 7901-NM cells transfected with a lentiviral vector encoding CD36 or negative control vector. K isotype IgG was used as a negative control. (B)3D spheroid BME cell invasion assay of SGC 7901-NM cells transfected with lentiviral vector encoding CD36 (LV-CD36) or with negative control vector (LV-NC). Photographs of all the spheroids in each well every 24 h for 7 days using a 4× objective. Quantitative analysis of the surface area of all spheroids. Normalized areas for all the spheroids are presented relative to the area on the first day. All of the areas were calculated three times using ImageJ, and the values represent the means ± SD. (C) The indicated cells were injected into nude mice (n = 6 for each group) via the tail vein along with weekly intraperitoneal injections of 20 μg of the anti-CD36 neutralizing monoclonal antibody JC63.1 or 20 μg of the corresponding IgA. Animals were sacrificed at 8 weeks after the injections. Photos of representative lung tissue samples in each group are shown. (D) Left: The histogram shows the proportion of mice with lung metastasis in each group. “Met” is short for “metastasis,” and “Met-free” indicates “metastasis-free.” Right: Mann Whitney test was used to evaluate the number of metastatic nodes in the lungs of mice from each group.

    Techniques Used: Flow Cytometry, Expressing, Transfection, Plasmid Preparation, Negative Control, Invasion Assay, Injection, Mouse Assay, MANN-WHITNEY

    11) Product Images from "A Booster Dose Enhances Immunogenicity of the COVID-19 Vaccine Candidate ChAdOx1 nCoV-19 in Aged Mice"

    Article Title: A Booster Dose Enhances Immunogenicity of the COVID-19 Vaccine Candidate ChAdOx1 nCoV-19 in Aged Mice

    Journal: Med (New York, N.y.)

    doi: 10.1016/j.medj.2020.12.006

    ChAdOx1 nCoV-19 Induces a Plasma Cell and Germinal Center B Cell Response (A) tSNE/FlowSOM analyses of CD19 + B cells from 3-month-old (3mo) mice 7 days after immunization with ChAdOx1 nCoV-19 or PBS; on the heatmap, red indicates high expression, and yellow indicates low expression. (B and C) Heatmaps of the manually gated B cell populations indicated at 7, 14, and 21 days after immunization in the iliac lymph node (B) and spleen (C); the gating strategy for these populations is shown in Figure S2 . Here, the frequency of each cell subset in each ChAdOx1 nCoV-19-immunized mouse has been expressed as the log2 fold change over the average frequency in PBS-immunized mice (n = 5). (D–J) Bar charts showing the total number of plasma cells (D), CD69 + IgD + B cells (E), proliferating non-germinal center B cells (F) and germinal center B cells (G) at the indicated time points after immunization. Number of T follicular helper (H) and T follicular regulatory cells (I) at the indicated time points post-immunization. Serum anti-spike IgM, IgG, and IgA (J) antibodies 7, 14, and 21 days after immunization. (K) Pie charts indicating the mean abundance of each IgG antibody subclass in the serum at the indicated time points after immunization. In (D)–(J), the bar height corresponds to the mean and each circle represents one biological replicate. p values are calculated using a Student’s t test with Holm-Sidak multiple testing correction; for ELISA, data analyses were done on log transformed values.
    Figure Legend Snippet: ChAdOx1 nCoV-19 Induces a Plasma Cell and Germinal Center B Cell Response (A) tSNE/FlowSOM analyses of CD19 + B cells from 3-month-old (3mo) mice 7 days after immunization with ChAdOx1 nCoV-19 or PBS; on the heatmap, red indicates high expression, and yellow indicates low expression. (B and C) Heatmaps of the manually gated B cell populations indicated at 7, 14, and 21 days after immunization in the iliac lymph node (B) and spleen (C); the gating strategy for these populations is shown in Figure S2 . Here, the frequency of each cell subset in each ChAdOx1 nCoV-19-immunized mouse has been expressed as the log2 fold change over the average frequency in PBS-immunized mice (n = 5). (D–J) Bar charts showing the total number of plasma cells (D), CD69 + IgD + B cells (E), proliferating non-germinal center B cells (F) and germinal center B cells (G) at the indicated time points after immunization. Number of T follicular helper (H) and T follicular regulatory cells (I) at the indicated time points post-immunization. Serum anti-spike IgM, IgG, and IgA (J) antibodies 7, 14, and 21 days after immunization. (K) Pie charts indicating the mean abundance of each IgG antibody subclass in the serum at the indicated time points after immunization. In (D)–(J), the bar height corresponds to the mean and each circle represents one biological replicate. p values are calculated using a Student’s t test with Holm-Sidak multiple testing correction; for ELISA, data analyses were done on log transformed values.

    Techniques Used: Mouse Assay, Expressing, Enzyme-linked Immunosorbent Assay, Transformation Assay

    12) Product Images from "Sulfur dioxide exposure reduces the quantity of CD19+ cells and causes nasal epithelial injury in rats"

    Article Title: Sulfur dioxide exposure reduces the quantity of CD19+ cells and causes nasal epithelial injury in rats

    Journal: Journal of Occupational Medicine and Toxicology (London, England)

    doi: 10.1186/s12995-018-0205-x

    The representative figure showed that SO 2 inhalation did not affect IgG levels in the serum and nasal septum. a : IgG level in the serum; b : IgG level in the nasal septum; c : IgE level in the serum; d : IgE level in the nasal septum; e : IgA level in the serum; f : IgA level in the nasal septum
    Figure Legend Snippet: The representative figure showed that SO 2 inhalation did not affect IgG levels in the serum and nasal septum. a : IgG level in the serum; b : IgG level in the nasal septum; c : IgE level in the serum; d : IgE level in the nasal septum; e : IgA level in the serum; f : IgA level in the nasal septum

    Techniques Used:

    13) Product Images from "In planta expression of nanobody-based designer chicken antibodies targeting Campylobacter"

    Article Title: In planta expression of nanobody-based designer chicken antibodies targeting Campylobacter

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0204222

    Screening for the expression of chimeric antibodies in A . thaliana ] were used. A His-tagged nanobody was used as a positive control, resulting in a protein band with a molecular weight of approximately 15 kDa. (A) IgA chimeric antibodies with V1, Nb23 and Nb5, (C) IgY chimeric antibodies with V1, Nb23 and Nb5.
    Figure Legend Snippet: Screening for the expression of chimeric antibodies in A . thaliana ] were used. A His-tagged nanobody was used as a positive control, resulting in a protein band with a molecular weight of approximately 15 kDa. (A) IgA chimeric antibodies with V1, Nb23 and Nb5, (C) IgY chimeric antibodies with V1, Nb23 and Nb5.

    Techniques Used: Expressing, Positive Control, Molecular Weight

    Expression of chimeric antibodies in transgenic A . thaliana seeds. Extracts of T2 seeds were coated and analysed by ELISA. The presence of chimeric antibodies in the extracts was tested, using anti-chicken IgA or IgY conjugated to HRP. As a negative control, extract of wild-type A . thaliana seeds was used. The results of seed extracts from A . thaliana plants transformed with Nb5-IgA, Nb23-IgA, V1-IgA, Nb5-IgY, Nb23-IgY and V1-IgY are shown.
    Figure Legend Snippet: Expression of chimeric antibodies in transgenic A . thaliana seeds. Extracts of T2 seeds were coated and analysed by ELISA. The presence of chimeric antibodies in the extracts was tested, using anti-chicken IgA or IgY conjugated to HRP. As a negative control, extract of wild-type A . thaliana seeds was used. The results of seed extracts from A . thaliana plants transformed with Nb5-IgA, Nb23-IgA, V1-IgA, Nb5-IgY, Nb23-IgY and V1-IgY are shown.

    Techniques Used: Expressing, Transgenic Assay, Enzyme-linked Immunosorbent Assay, Negative Control, Transformation Assay

    Visualisation of the binding of chimeric anti-MOMP antibodies in seed extract with Campylobacter isolates. (A, B, C, J, K, L) C . jejuni strain KC40, (D, E, F, M, N, O) C . jejuni strain Cam12/0156 and (G, H, I, P, Q, R) C . coli strain K43/5. Binding with the different Campylobacter isolates is shown with seed extract containing (A, D, G) Nb5-IgA, (B, E, H) Nb23-IgA, (C, F, I) V1-IgA, (J, M, P) Nb5-IgY, (K, N, Q) Nb23-IgY and (L, O, R) V1-IgY. As a negative control, the nanobody V1 against F4-fimbriated E . coli was used. Bright field microscopy was used for the visualisation of the corresponding bacterial cells.
    Figure Legend Snippet: Visualisation of the binding of chimeric anti-MOMP antibodies in seed extract with Campylobacter isolates. (A, B, C, J, K, L) C . jejuni strain KC40, (D, E, F, M, N, O) C . jejuni strain Cam12/0156 and (G, H, I, P, Q, R) C . coli strain K43/5. Binding with the different Campylobacter isolates is shown with seed extract containing (A, D, G) Nb5-IgA, (B, E, H) Nb23-IgA, (C, F, I) V1-IgA, (J, M, P) Nb5-IgY, (K, N, Q) Nb23-IgY and (L, O, R) V1-IgY. As a negative control, the nanobody V1 against F4-fimbriated E . coli was used. Bright field microscopy was used for the visualisation of the corresponding bacterial cells.

    Techniques Used: Binding Assay, Negative Control, Microscopy

    Binding of chimeric antibodies to C . jejuni KC40 bacteria and purified MOMP. KC40 bacteria and MOMP (1 μ g/ml) were coated in an ELISA plate. Subsequently, the interaction of twofold serial dilutions of the seed extracts was assessed. Therefore, anti-IgA and anti-IgY antibodies, conjugated to HRP, were used for the detection of chimeric antibodies, bound to the bacteria or to the purified native MOMP antigen. As a negative control, the binding of extracts of wild-type A . thaliana seeds with the bacteria and MOMP was measured. (A) Nb5-IgA, (B) Nb23-IgA, (C) Nb5-IgY and (D) Nb23-IgY. The error bars correspond to the standard deviations.
    Figure Legend Snippet: Binding of chimeric antibodies to C . jejuni KC40 bacteria and purified MOMP. KC40 bacteria and MOMP (1 μ g/ml) were coated in an ELISA plate. Subsequently, the interaction of twofold serial dilutions of the seed extracts was assessed. Therefore, anti-IgA and anti-IgY antibodies, conjugated to HRP, were used for the detection of chimeric antibodies, bound to the bacteria or to the purified native MOMP antigen. As a negative control, the binding of extracts of wild-type A . thaliana seeds with the bacteria and MOMP was measured. (A) Nb5-IgA, (B) Nb23-IgA, (C) Nb5-IgY and (D) Nb23-IgY. The error bars correspond to the standard deviations.

    Techniques Used: Binding Assay, Purification, Enzyme-linked Immunosorbent Assay, Negative Control

    Transient expression of chimeric antibodies carrying Nb5 in leaves of N . benthamiana . Protein extracts were analysed using (A) SDS-PAGE stained with Coomassie blue dye and (B) western blot developed with mouse anti-histidine tag monoclonal antibody. Extracts of N . benthamiana leaves transformed with Nb5-IgA and Nb5-IgY fusion constructs were tested. Leaves infiltrated with A . tumefaciens harbouring the vector pEAQ -HT were used as negative control.
    Figure Legend Snippet: Transient expression of chimeric antibodies carrying Nb5 in leaves of N . benthamiana . Protein extracts were analysed using (A) SDS-PAGE stained with Coomassie blue dye and (B) western blot developed with mouse anti-histidine tag monoclonal antibody. Extracts of N . benthamiana leaves transformed with Nb5-IgA and Nb5-IgY fusion constructs were tested. Leaves infiltrated with A . tumefaciens harbouring the vector pEAQ -HT were used as negative control.

    Techniques Used: Expressing, SDS Page, Staining, Western Blot, Transformation Assay, Construct, Plasmid Preparation, Negative Control

    Interaction of chimeric antibodies in seed extract from homozygous plants with their antigen. SDS-PAGE and a western blot were performed on purified MOMP under non-denaturing conditions. (A) SDS-PAGE with purified native MOMP stained with Coomassie blue dye. The results of the western blot confirm the interactions of (B) Nb23-IgA14D and (C) Nb23-IgY12C, with MOMP. (D) Wild-type extract was used as a negative control. The western blot was developed with anti-IgA or anti-IgY antibodies conjugated to HRP.
    Figure Legend Snippet: Interaction of chimeric antibodies in seed extract from homozygous plants with their antigen. SDS-PAGE and a western blot were performed on purified MOMP under non-denaturing conditions. (A) SDS-PAGE with purified native MOMP stained with Coomassie blue dye. The results of the western blot confirm the interactions of (B) Nb23-IgA14D and (C) Nb23-IgY12C, with MOMP. (D) Wild-type extract was used as a negative control. The western blot was developed with anti-IgA or anti-IgY antibodies conjugated to HRP.

    Techniques Used: SDS Page, Western Blot, Purification, Staining, Negative Control

    14) Product Images from "Subunit Nanovaccine with Potent Cellular and Mucosal Immunity for COVID-19"

    Article Title: Subunit Nanovaccine with Potent Cellular and Mucosal Immunity for COVID-19

    Journal: ACS Applied Bio Materials

    doi: 10.1021/acsabm.0c00668

    Nanovaccines elicit strong humoral immunity. (A) Schematic of the immunization strategy. (B) Quantitative comparison of the S1-specific production of IgG, IgG1, IgG2a and IgG2a/IgG1 on day 42. (C) Change of antibody titer over time (* represents the comparison of p(M+C)–S1 with f(M+C+S1); # represents the comparison of p(M+C)–S1 with A+S1), n = 10. (D) Quantitative comparison of the S1-specific production of IgA at day 65. Data are presented as the mean ± SEM (* P or # P
    Figure Legend Snippet: Nanovaccines elicit strong humoral immunity. (A) Schematic of the immunization strategy. (B) Quantitative comparison of the S1-specific production of IgG, IgG1, IgG2a and IgG2a/IgG1 on day 42. (C) Change of antibody titer over time (* represents the comparison of p(M+C)–S1 with f(M+C+S1); # represents the comparison of p(M+C)–S1 with A+S1), n = 10. (D) Quantitative comparison of the S1-specific production of IgA at day 65. Data are presented as the mean ± SEM (* P or # P

    Techniques Used:

    15) Product Images from "High APRIL Levels Are Associated With Slow Disease Progression and Low Immune Activation in Chronic HIV-1-Infected Patients"

    Article Title: High APRIL Levels Are Associated With Slow Disease Progression and Low Immune Activation in Chronic HIV-1-Infected Patients

    Journal: Frontiers in Medicine

    doi: 10.3389/fmed.2020.00299

    Associations between the plasma levels of APRIL and immunoglobulin production. (A) Associations between the APRIL levels and HIV-1-specific IgM in HIV-1-infected individuals ( n = 59). (B) Associations between the APRIL levels and HIV-1-specific IgA in HIV-1-infected individuals ( n = 59). (C) Associations between the APRIL levels and neutralization titers in untreated HIV-1-infected individuals ( n = 46). (D) Associations between the APRIL levels and neutralization breadth in untreated HIV-1-infected individuals ( n = 46). LTNPs-B, long-term non-progressors at baseline; LTNPs-L, long-term non-progressors at the latest measurement; TPs, typical progressors; ART, antiretroviral therapy; ID 50 , 50% inhibitory dilution. Intergroup comparisons were performed using a Kruskal–Wallis test, followed by Dunn's post-test; p and r values were calculated by Spearman's rank correlation tests (* p
    Figure Legend Snippet: Associations between the plasma levels of APRIL and immunoglobulin production. (A) Associations between the APRIL levels and HIV-1-specific IgM in HIV-1-infected individuals ( n = 59). (B) Associations between the APRIL levels and HIV-1-specific IgA in HIV-1-infected individuals ( n = 59). (C) Associations between the APRIL levels and neutralization titers in untreated HIV-1-infected individuals ( n = 46). (D) Associations between the APRIL levels and neutralization breadth in untreated HIV-1-infected individuals ( n = 46). LTNPs-B, long-term non-progressors at baseline; LTNPs-L, long-term non-progressors at the latest measurement; TPs, typical progressors; ART, antiretroviral therapy; ID 50 , 50% inhibitory dilution. Intergroup comparisons were performed using a Kruskal–Wallis test, followed by Dunn's post-test; p and r values were calculated by Spearman's rank correlation tests (* p

    Techniques Used: Infection, Neutralization

    16) Product Images from "Alternative antibiotic feed additives alleviate pneumonia with inhibiting ACE‐2 expression in the respiratory system of piglets, et al. Alternative antibiotic feed additives alleviate pneumonia with inhibiting ACE‐2 expression in the respiratory system of piglets"

    Article Title: Alternative antibiotic feed additives alleviate pneumonia with inhibiting ACE‐2 expression in the respiratory system of piglets, et al. Alternative antibiotic feed additives alleviate pneumonia with inhibiting ACE‐2 expression in the respiratory system of piglets

    Journal: Food Science & Nutrition

    doi: 10.1002/fsn3.2089

    GLZ treatment alleviates epithelial cilia shedding and pneumonia and increases immunity in weanling piglets. (a) Representative H E stains of trachea, bronchus, and lung of weanling piglets with sham, TAB, and GLZ treatments at the 10th week of age. Epithelial cilia were fragmentary in the tracheal and bronchial tissues of the sham group but were complete in the TAB and GLZ groups (marked with arrows); scale bar = 30 μm. In the lung tissue, pneumonia was severe in the sham and TAB groups but was not observed in the GLZ group; scale bar = 150 μm. Enlarged views of the lung tissue are shown in the lower left corner; scale bars = 30 μm. (b) Comparisons of IgA, IgG, and IgE in the blood of weanling piglets with sham, TAB, and GLZ treatments at the 10th week of age. Weanling piglets with GLZ treatment showed significantly higher IgA and IgG but significantly lower IgE than those with sham and TAB treatments. All data are shown as mean ± standard error of mean ( SEM ) (** p
    Figure Legend Snippet: GLZ treatment alleviates epithelial cilia shedding and pneumonia and increases immunity in weanling piglets. (a) Representative H E stains of trachea, bronchus, and lung of weanling piglets with sham, TAB, and GLZ treatments at the 10th week of age. Epithelial cilia were fragmentary in the tracheal and bronchial tissues of the sham group but were complete in the TAB and GLZ groups (marked with arrows); scale bar = 30 μm. In the lung tissue, pneumonia was severe in the sham and TAB groups but was not observed in the GLZ group; scale bar = 150 μm. Enlarged views of the lung tissue are shown in the lower left corner; scale bars = 30 μm. (b) Comparisons of IgA, IgG, and IgE in the blood of weanling piglets with sham, TAB, and GLZ treatments at the 10th week of age. Weanling piglets with GLZ treatment showed significantly higher IgA and IgG but significantly lower IgE than those with sham and TAB treatments. All data are shown as mean ± standard error of mean ( SEM ) (** p

    Techniques Used:

    17) Product Images from "Glycoconjugate Vaccine Containing Escherichia coli O157:H7 O-Antigen Linked with Maltose-Binding Protein Elicits Humoral and Cellular Responses"

    Article Title: Glycoconjugate Vaccine Containing Escherichia coli O157:H7 O-Antigen Linked with Maltose-Binding Protein Elicits Humoral and Cellular Responses

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0105215

    Antibody responses after third immunization. ( A ), E. coli O157:H7-specific IgG antibody titers of serum samples; ( B ), E. coli O157:H7-specific IgM antibody titers of serum samples; ( C ), E. coli O157:H7-specific IgA antibody titers of feces samples; ( D ), MBP-specific IgG antibody titers of serum samples. The cutoff value was OD negative control ×2.1. Results were expressed as the arithmetic mean ±SD indicated by error bars. Differences of two groups were indicated with symbols (*: P
    Figure Legend Snippet: Antibody responses after third immunization. ( A ), E. coli O157:H7-specific IgG antibody titers of serum samples; ( B ), E. coli O157:H7-specific IgM antibody titers of serum samples; ( C ), E. coli O157:H7-specific IgA antibody titers of feces samples; ( D ), MBP-specific IgG antibody titers of serum samples. The cutoff value was OD negative control ×2.1. Results were expressed as the arithmetic mean ±SD indicated by error bars. Differences of two groups were indicated with symbols (*: P

    Techniques Used: Negative Control

    18) Product Images from "TLR7 in B cells promotes renal inflammation and Gd-IgA1 synthesis in IgA nephropathy"

    Article Title: TLR7 in B cells promotes renal inflammation and Gd-IgA1 synthesis in IgA nephropathy

    Journal: JCI Insight

    doi: 10.1172/jci.insight.136965

    TLR7-GALNT2 axis affected galactose deficiency of IgA1 antibodies in B cells. ( A and B ) Synthetic siRNA targeting human TLR7 gene or control siRNA were electroporated into EBV-immortalized B cell lines derived from patients with IgAN ( A ) and DAKIKI B cell lines ( B ). siNC, negative control; siTLR7, siRNA for TLR7 ; siScramble, scramble siRNA control for TLR7 . ( C ) Adenoviruses carrying coding sequences for GALNT2 (AdGALNT2) or GFP (AdGFP) gene were transfected into EBV-immortalized B cells derived from patients with IgAN. Cells were collected after 72 hours and analyzed by Western blot for target proteins. These experiments were repeated independently 3 times, and representative data was selected for display. ( D ) Culture supernatant was collected 6 days after transfection and analyzed by IgA1 and Gd-gA1 ELISA ( n = 7). Data are presented as mean ± SEM and analyzed by 2-tailed unpaired Student’s t test. *** P
    Figure Legend Snippet: TLR7-GALNT2 axis affected galactose deficiency of IgA1 antibodies in B cells. ( A and B ) Synthetic siRNA targeting human TLR7 gene or control siRNA were electroporated into EBV-immortalized B cell lines derived from patients with IgAN ( A ) and DAKIKI B cell lines ( B ). siNC, negative control; siTLR7, siRNA for TLR7 ; siScramble, scramble siRNA control for TLR7 . ( C ) Adenoviruses carrying coding sequences for GALNT2 (AdGALNT2) or GFP (AdGFP) gene were transfected into EBV-immortalized B cells derived from patients with IgAN. Cells were collected after 72 hours and analyzed by Western blot for target proteins. These experiments were repeated independently 3 times, and representative data was selected for display. ( D ) Culture supernatant was collected 6 days after transfection and analyzed by IgA1 and Gd-gA1 ELISA ( n = 7). Data are presented as mean ± SEM and analyzed by 2-tailed unpaired Student’s t test. *** P

    Techniques Used: Derivative Assay, Negative Control, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay

    Secretion of IgA1 and Gd-IgA1 was enhanced in PBMCs of patients with IgAN via activation of TLR7/8 in vitro. ( A ) PBMCs from healthy controls (HC, n = 72), patients with IgAN (IgAN, n = 49), and disease controls (DC, n = 29) were purified and cultured with R848 (5 μg/mL) for 12 days. Culture supernatant was collected and measured for IgA1 content and IgA1 galactose deficiency by sandwich ELISA and HAA lectin binding assay, respectively. One-way ANOVA with Bonferroni’s post hoc test. * P
    Figure Legend Snippet: Secretion of IgA1 and Gd-IgA1 was enhanced in PBMCs of patients with IgAN via activation of TLR7/8 in vitro. ( A ) PBMCs from healthy controls (HC, n = 72), patients with IgAN (IgAN, n = 49), and disease controls (DC, n = 29) were purified and cultured with R848 (5 μg/mL) for 12 days. Culture supernatant was collected and measured for IgA1 content and IgA1 galactose deficiency by sandwich ELISA and HAA lectin binding assay, respectively. One-way ANOVA with Bonferroni’s post hoc test. * P

    Techniques Used: Activation Assay, In Vitro, Purification, Cell Culture, Sandwich ELISA, Binding Assay

    Increased expression of TLR7 in peripheral B cells of patients with IgAN. ( A ) The relative mRNA levels of TLRs and O-glycosyltransferases in PBMCs. PBMCs from patients with IgAN ( n = 46), healthy controls ( n = 40), and disease donors ( n = 26) were prepared by Ficoll-Paque density centrifugation and subjected to quantitative PCR analysis for target genes, using GAPDH as internal controls. Statistical significance was determined by 1-way ANOVA with Bonferroni’s correction. ( B ) The association analysis of relative mRNA levels of TLR7 and GALNT2 in PBMCs of IgAN patients. ( C ) The relative mRNA levels of TLRs and O-glycosyltransferases in sorted fresh CD19 + B cells from patients with IgAN ( n = 6–10) and healthy controls ( n = 5–8). ( D ) The expression of TLR7, GALNT2, and IgA proteins in EBV-immortalized B cells from patients with IgAN ( n = 11) and healthy donors ( n = 6), as detected by Western blot. ( E ) Quantification and statistical data for D , using GAPDH as internal control. ( F ) The association analysis of relative protein levels of TLR7 and GALNT2 in EBV-immortalized B cells derived from IgAN patients. HC, healthy controls; IgAN, patients with IgAN; DC, disease controls (MCD/MN). Data were presented as median ± SEM and analyzed by 2-tailed unpaired Student’s t test ( C and E ) and Pearson correlation analysis ( B and F ). * P
    Figure Legend Snippet: Increased expression of TLR7 in peripheral B cells of patients with IgAN. ( A ) The relative mRNA levels of TLRs and O-glycosyltransferases in PBMCs. PBMCs from patients with IgAN ( n = 46), healthy controls ( n = 40), and disease donors ( n = 26) were prepared by Ficoll-Paque density centrifugation and subjected to quantitative PCR analysis for target genes, using GAPDH as internal controls. Statistical significance was determined by 1-way ANOVA with Bonferroni’s correction. ( B ) The association analysis of relative mRNA levels of TLR7 and GALNT2 in PBMCs of IgAN patients. ( C ) The relative mRNA levels of TLRs and O-glycosyltransferases in sorted fresh CD19 + B cells from patients with IgAN ( n = 6–10) and healthy controls ( n = 5–8). ( D ) The expression of TLR7, GALNT2, and IgA proteins in EBV-immortalized B cells from patients with IgAN ( n = 11) and healthy donors ( n = 6), as detected by Western blot. ( E ) Quantification and statistical data for D , using GAPDH as internal control. ( F ) The association analysis of relative protein levels of TLR7 and GALNT2 in EBV-immortalized B cells derived from IgAN patients. HC, healthy controls; IgAN, patients with IgAN; DC, disease controls (MCD/MN). Data were presented as median ± SEM and analyzed by 2-tailed unpaired Student’s t test ( C and E ) and Pearson correlation analysis ( B and F ). * P

    Techniques Used: Expressing, Centrifugation, Real-time Polymerase Chain Reaction, Western Blot, Derivative Assay

    19) Product Images from "Development of disease and immunity at the genital epithelium following intrarectal inoculation of male guinea pigs with Herpes simplex virus type 2"

    Article Title: Development of disease and immunity at the genital epithelium following intrarectal inoculation of male guinea pigs with Herpes simplex virus type 2

    Journal: Virology

    doi: 10.1016/j.virol.2018.10.023

    Isotypes of HSV-specific serum antibodies following IREC HSV-2 inoculation of male guinea pigs. Serum was collected from male guinea pigs between 1.5- 4.5 months after inoculation and the endpoint titer of IgA ( A ), IgG ( B ), IgG1 ( C ), and IgG2 ( D ) gD-specific antibodies was determined by ELISA. Results shown are the mean endpoint titer ± SEM for 22 HSV-2 inoculated guinea pigs from three separate experiments. The limit of detection for these assays was a 1:10 dilution. Asterisk indicates significant differences between endpoint titers of sera from immune and naïve animals (P
    Figure Legend Snippet: Isotypes of HSV-specific serum antibodies following IREC HSV-2 inoculation of male guinea pigs. Serum was collected from male guinea pigs between 1.5- 4.5 months after inoculation and the endpoint titer of IgA ( A ), IgG ( B ), IgG1 ( C ), and IgG2 ( D ) gD-specific antibodies was determined by ELISA. Results shown are the mean endpoint titer ± SEM for 22 HSV-2 inoculated guinea pigs from three separate experiments. The limit of detection for these assays was a 1:10 dilution. Asterisk indicates significant differences between endpoint titers of sera from immune and naïve animals (P

    Techniques Used: Enzyme-linked Immunosorbent Assay

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    Article Title: Cysteines and N-Glycosylation Sites Conserved among All Alphaherpesviruses Regulate Membrane Fusion in Herpes Simplex Virus 1 Infection
    Article Snippet: Immunoblot assays were carried out using anti-VP5 (ab6508; Abcam), rabbit anti-beta tubulin (ab179513; Abcam), goat anti-mouse HRP (Abcam), and goat anti-rabbit HRP (Abcam).

    Article Title: Nutrient-Deprived Retinal Progenitors Proliferate in Response to Hypoxia: Interaction of the HIF-1 and mTOR Pathway
    Article Snippet: For detection, rabbit anti-HIF-1 (1:500, ab2185 Abcam), rabbit anti-pS6 Ser235/236 (1:1000, Cell Signalling), mouse anti-α-tubulin (1:5000, Abcam), goat anti-rabbit HRP-conjugated (1:1000 Abcam) and goat anti-mouse HRP-conjugated (1:1000; Abcam) antibodies were used.

    Incubation:

    Article Title: Recombinant Mycobacterium smegmatis with a pMyong2 vector expressing Human Immunodeficiency Virus Type I Gag can induce enhanced virus-specific immune responses
    Article Snippet: Mouse anti-p24 monoclonal antibody (Abcam, Cambridge, USA; 1:1,000 dilution) was added and incubated overnight at 4 °C. .. After incubation, the membranes were treated with the HRP-conjugated goat anti-mouse secondary antibody (Abcam, 1:2,000 dilution) for 1 hr at room temperature. ..

    Article Title: MTBP promotes migration and invasion by regulation of ZEB2-mediated epithelial–mesenchymal transition in lung cancer cells
    Article Snippet: Membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 for 1 hour at room temperature and then incubated with the following specified antibodies for 1 hour at room temperature: anti-MTBP (1:2,000 dilution; Sigma-Aldrich), anti-ZEB2 (1:5,000 dilution; Abcam), anti-E-cadherin (1:5,000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-N-cadherin (1:5,000 dilution; Cell Signaling Technology), anti-β-catenin (1:5,000 dilution; Cell Signaling Technology), anti-Vimentin (1:5,000 dilution; Cell Signaling Technology), and anti-GAPDH (1:5,000 dilution; Cell Signaling Technology). .. The membranes were then incubated with HRP-conjugated goat anti-mouse or anti-rabbit IgG (ZSGB-BIO) for 1 hour at room temperature. .. Signals were visualized using an enhanced chemiluminescence reagent and captured using AI600 version 1.2.0 on Amersham Imager 600 (GE Healthcare, Chicago, IL, USA).

    Article Title: Notch signaling contributes to the expression of inflammatory cytokines induced by highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) infection in porcine alveolar macrophages
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    Western Blot:

    Article Title: Hydrogen peroxide induces Arl1 degradation and impairs Golgi-mediated trafficking
    Article Snippet: Secondary antibodies used for fluorescence microscopy include fluorescence-labeled goat anti-mouse, goat anti-rabbit, and goat anti-sheep antibodies. .. Secondary antibodies used for Western blot include HRP-conjugated goat anti-mouse and goat anti-rabbit antibodies. .. Cell culture and drug treatments HeLa were obtained from ATCC (Manassas, VA), cultured in DMEM (ThermoFisher) supplemented with 10% fetal bovine serum (Gemini Bio-Products, Sacramento, CA), and 100 units/ml penicillin–streptomycin at 37°C with 5% CO2 , and routinely screened for mycoplasma contamination.

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    Abcam goat anti human α sma antibody
    Multiple mechanisms decreasing HIFα in sulfoquinovosyl-acylpropanediol (SQAP). ( a ) The presence of the proteasome inhibitor MG-132 decreased the effect of SQAP on HIFα protein levels. HAK1-B cells incubated under either normoxia or 3% O 2 hypoxia were treated with 10 μmo/l SQAP for 24 hours and further incubated for 4 hours in the presence of 10 μmo/l MG-132. ( b ) HIF1α synthesis at the transcriptional level decreased upon SQAP addition in a dose-dependent manner. HAK-1B cells were incubated with medium containing different dose of SQAP (1 and 10 μmo/l). Cellular RNA was then extracted and analyzed for HIF1α mRNA expression by quantitative real-time polymerase chain reaction, with GAPDH as a control. ( c ) SQAP decreases NFκB expression in HAK1-B and Huh-7. Western blots for both cell lines exposed to SQAP (1 and 10 μmo/l) under hypoxic conditions are shown. Cells were incubated with SQAP-containing medium for 24 hours, after which the cells were moved to 3% O 2 hypoxic conditions for 24 hours and then lysed with radioimmunoprecipitation buffer. ( d ) Representative immunohistochemistry images for pimonidazole in HAK1-B tumor treated with SQAP. Scale bar = 200 μm. ( e ) Western blot analysis of pimonidazole adducts in HAK1-B tumors treated with SQAP. Whole tumors were collected from tumor bearing mice ( n = 4 per group). Negative control: HAK1-B cells cultured under normoxia in vitro . Positive control: HAK1-B cell cultured under 3% O 2 hypoxia in vitro . Pimonidazole adducts are shown in the indicated multiple bands. The band densities for each protein were measured and calibrated by β-actin. Average bands densities are represented as mean ± standard deviation (SD). ( f ) Representative double immunofluorescence images using both CD31 (green) and <t>α-SMA</t> (red) in HAK1-B tumors treated with SQAP. Nuclei were stained with DAPI. Scale bar = 50 μm. Relative number of tumor vessels, α-SMA-positive cells, and tumor vessels covered with α-SMA positive cells in HAK1-B tumors treated with SQAP are shown. The number of tumor vessels and the ratio of pericyte covered vessels are represented as mean ± SD. DAPI, 4’,6-diamidino-2-phenylindole, dihydrochloride.
    Goat Anti Human α Sma Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam mouse monoclonal anti α subunit
    Distribution and expression changes of the <t>α-subunit</t> of the calcium-activated potassium channels ( K Ca channels) in the tumor tissues from the different groups (scale bar = 20 um). (A) Distribution and expression of the α-subunit of the K Ca channels by immunohistochemistry. (B) The α-subunit of the K Ca channels protein expression changes by a Western blot analysis in the tumors from the different groups. The quantification of the α-subunit of the K Ca channels expression was performed by scanning the intensity of the densitometry value ( n = 5 for each group; values represent the mean ± standard deviation). (C) The α-subunit of the K Ca channels mRNA expression changes by a qRT-PCR analysis in the tumors from the different groups ( n = 5 for each group; values represent the mean ± standard deviation). * Indicates a significant difference compared with the control group ( p
    Mouse Monoclonal Anti α Subunit, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit anti human α sma
    IL-17A induces proliferation, collagen synthesis and secretion of SSc patient-derived DVSMCs via ERK1/2 signing pathway. (A) DVSMCs were pre-treated with PD 98059 (10 μM/ml) for 2 h before incubation with IL-17A at 100 ng/ml for 24 h, 48 h, 72 h, and cell proliferation was tested using cell counting kit-8. (B) The cells were pre-treated with PD98059 for 2 h before incubation with the serum of SSc patients or healthy individuals for 24 h, 48 h, 72 h, and cell proliferation was tested using CCK8. (C) The cells were pre-treated with PD 98059 for 2 h before incubation with IL-17A at 100 ng/ml for 24 h. The activity of collagen1α1 or collagen3α1 proximal promoter was detected using a dual luciferase reporter gene assay. (D) After being pre-treated with PD98059 for 2h before incubation with the serum of SSc patients or healthy individuals for 24 h, the activity of collagen1α1 or collagen3α1 proximal promoter was also detected. Data were represented as mean ratios of Firefly to Renilla luciferase activity. (E, F) The cells were pre-treated with PD 98059 for 2 h before incubation with IL-17A or SSc serum for 24 h, the gene expression of collagen 1 and collagen 3 was measured using real-time RT-PCR analysis. (G, H) The cells were pre-treated with PD 98059 for 2 h before incubation with IL-17A or SSc serum for 24 h, the concentration of collagen 1, collagen 3 was detected using ELISA. (I, J) The cells were pre-treated with PD98059 for 2 h before incubation with IL-17A, serum from SSc patients and healthy individuals for 24 h, and the protein expressions of collagen 1, collagen 3, IL-17RA and <t>α-SMA</t> were measured using Western blot. GAPDH was used as a loading control. The experiment was repeated three times, and the data are presented as means ± standard deviation.
    Rabbit Anti Human α Sma, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Multiple mechanisms decreasing HIFα in sulfoquinovosyl-acylpropanediol (SQAP). ( a ) The presence of the proteasome inhibitor MG-132 decreased the effect of SQAP on HIFα protein levels. HAK1-B cells incubated under either normoxia or 3% O 2 hypoxia were treated with 10 μmo/l SQAP for 24 hours and further incubated for 4 hours in the presence of 10 μmo/l MG-132. ( b ) HIF1α synthesis at the transcriptional level decreased upon SQAP addition in a dose-dependent manner. HAK-1B cells were incubated with medium containing different dose of SQAP (1 and 10 μmo/l). Cellular RNA was then extracted and analyzed for HIF1α mRNA expression by quantitative real-time polymerase chain reaction, with GAPDH as a control. ( c ) SQAP decreases NFκB expression in HAK1-B and Huh-7. Western blots for both cell lines exposed to SQAP (1 and 10 μmo/l) under hypoxic conditions are shown. Cells were incubated with SQAP-containing medium for 24 hours, after which the cells were moved to 3% O 2 hypoxic conditions for 24 hours and then lysed with radioimmunoprecipitation buffer. ( d ) Representative immunohistochemistry images for pimonidazole in HAK1-B tumor treated with SQAP. Scale bar = 200 μm. ( e ) Western blot analysis of pimonidazole adducts in HAK1-B tumors treated with SQAP. Whole tumors were collected from tumor bearing mice ( n = 4 per group). Negative control: HAK1-B cells cultured under normoxia in vitro . Positive control: HAK1-B cell cultured under 3% O 2 hypoxia in vitro . Pimonidazole adducts are shown in the indicated multiple bands. The band densities for each protein were measured and calibrated by β-actin. Average bands densities are represented as mean ± standard deviation (SD). ( f ) Representative double immunofluorescence images using both CD31 (green) and α-SMA (red) in HAK1-B tumors treated with SQAP. Nuclei were stained with DAPI. Scale bar = 50 μm. Relative number of tumor vessels, α-SMA-positive cells, and tumor vessels covered with α-SMA positive cells in HAK1-B tumors treated with SQAP are shown. The number of tumor vessels and the ratio of pericyte covered vessels are represented as mean ± SD. DAPI, 4’,6-diamidino-2-phenylindole, dihydrochloride.

    Journal: Molecular Therapy Oncolytics

    Article Title: Inhibition of hypoxia-inducible factor via upregulation of von Hippel-Lindau protein induces “angiogenic switch off” in a hepatoma mouse model

    doi: 10.1038/mto.2015.20

    Figure Lengend Snippet: Multiple mechanisms decreasing HIFα in sulfoquinovosyl-acylpropanediol (SQAP). ( a ) The presence of the proteasome inhibitor MG-132 decreased the effect of SQAP on HIFα protein levels. HAK1-B cells incubated under either normoxia or 3% O 2 hypoxia were treated with 10 μmo/l SQAP for 24 hours and further incubated for 4 hours in the presence of 10 μmo/l MG-132. ( b ) HIF1α synthesis at the transcriptional level decreased upon SQAP addition in a dose-dependent manner. HAK-1B cells were incubated with medium containing different dose of SQAP (1 and 10 μmo/l). Cellular RNA was then extracted and analyzed for HIF1α mRNA expression by quantitative real-time polymerase chain reaction, with GAPDH as a control. ( c ) SQAP decreases NFκB expression in HAK1-B and Huh-7. Western blots for both cell lines exposed to SQAP (1 and 10 μmo/l) under hypoxic conditions are shown. Cells were incubated with SQAP-containing medium for 24 hours, after which the cells were moved to 3% O 2 hypoxic conditions for 24 hours and then lysed with radioimmunoprecipitation buffer. ( d ) Representative immunohistochemistry images for pimonidazole in HAK1-B tumor treated with SQAP. Scale bar = 200 μm. ( e ) Western blot analysis of pimonidazole adducts in HAK1-B tumors treated with SQAP. Whole tumors were collected from tumor bearing mice ( n = 4 per group). Negative control: HAK1-B cells cultured under normoxia in vitro . Positive control: HAK1-B cell cultured under 3% O 2 hypoxia in vitro . Pimonidazole adducts are shown in the indicated multiple bands. The band densities for each protein were measured and calibrated by β-actin. Average bands densities are represented as mean ± standard deviation (SD). ( f ) Representative double immunofluorescence images using both CD31 (green) and α-SMA (red) in HAK1-B tumors treated with SQAP. Nuclei were stained with DAPI. Scale bar = 50 μm. Relative number of tumor vessels, α-SMA-positive cells, and tumor vessels covered with α-SMA positive cells in HAK1-B tumors treated with SQAP are shown. The number of tumor vessels and the ratio of pericyte covered vessels are represented as mean ± SD. DAPI, 4’,6-diamidino-2-phenylindole, dihydrochloride.

    Article Snippet: After blocking, sections were incubated with goat anti-human α-SMA antibody (1:100; Abcam) at 4 °C overnight and Alexa fluor 568-labeled anti-goat IgG antibody (1:100; Invitrogen) for 1 hour.

    Techniques: Incubation, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Mouse Assay, Negative Control, Cell Culture, In Vitro, Positive Control, Standard Deviation, Immunofluorescence, Staining

    Distribution and expression changes of the α-subunit of the calcium-activated potassium channels ( K Ca channels) in the tumor tissues from the different groups (scale bar = 20 um). (A) Distribution and expression of the α-subunit of the K Ca channels by immunohistochemistry. (B) The α-subunit of the K Ca channels protein expression changes by a Western blot analysis in the tumors from the different groups. The quantification of the α-subunit of the K Ca channels expression was performed by scanning the intensity of the densitometry value ( n = 5 for each group; values represent the mean ± standard deviation). (C) The α-subunit of the K Ca channels mRNA expression changes by a qRT-PCR analysis in the tumors from the different groups ( n = 5 for each group; values represent the mean ± standard deviation). * Indicates a significant difference compared with the control group ( p

    Journal: Frontiers in Neuroscience

    Article Title: Increasing of Blood-Brain Tumor Barrier Permeability through Transcellular and Paracellular Pathways by Microbubble-Enhanced Diagnostic Ultrasound in a C6 Glioma Model

    doi: 10.3389/fnins.2017.00086

    Figure Lengend Snippet: Distribution and expression changes of the α-subunit of the calcium-activated potassium channels ( K Ca channels) in the tumor tissues from the different groups (scale bar = 20 um). (A) Distribution and expression of the α-subunit of the K Ca channels by immunohistochemistry. (B) The α-subunit of the K Ca channels protein expression changes by a Western blot analysis in the tumors from the different groups. The quantification of the α-subunit of the K Ca channels expression was performed by scanning the intensity of the densitometry value ( n = 5 for each group; values represent the mean ± standard deviation). (C) The α-subunit of the K Ca channels mRNA expression changes by a qRT-PCR analysis in the tumors from the different groups ( n = 5 for each group; values represent the mean ± standard deviation). * Indicates a significant difference compared with the control group ( p

    Article Snippet: Then, the sections were incubated with a rabbit polyclonal anti-JAM-A antibody (Abcam, Cambridge, UK) and a mouse monoclonal anti-α-subunit of K Ca channels antibody (Abcam, Cambridge, UK) at 4°C overnight.

    Techniques: Expressing, Immunohistochemistry, Western Blot, Standard Deviation, Quantitative RT-PCR

    IL-17A induces proliferation, collagen synthesis and secretion of SSc patient-derived DVSMCs via ERK1/2 signing pathway. (A) DVSMCs were pre-treated with PD 98059 (10 μM/ml) for 2 h before incubation with IL-17A at 100 ng/ml for 24 h, 48 h, 72 h, and cell proliferation was tested using cell counting kit-8. (B) The cells were pre-treated with PD98059 for 2 h before incubation with the serum of SSc patients or healthy individuals for 24 h, 48 h, 72 h, and cell proliferation was tested using CCK8. (C) The cells were pre-treated with PD 98059 for 2 h before incubation with IL-17A at 100 ng/ml for 24 h. The activity of collagen1α1 or collagen3α1 proximal promoter was detected using a dual luciferase reporter gene assay. (D) After being pre-treated with PD98059 for 2h before incubation with the serum of SSc patients or healthy individuals for 24 h, the activity of collagen1α1 or collagen3α1 proximal promoter was also detected. Data were represented as mean ratios of Firefly to Renilla luciferase activity. (E, F) The cells were pre-treated with PD 98059 for 2 h before incubation with IL-17A or SSc serum for 24 h, the gene expression of collagen 1 and collagen 3 was measured using real-time RT-PCR analysis. (G, H) The cells were pre-treated with PD 98059 for 2 h before incubation with IL-17A or SSc serum for 24 h, the concentration of collagen 1, collagen 3 was detected using ELISA. (I, J) The cells were pre-treated with PD98059 for 2 h before incubation with IL-17A, serum from SSc patients and healthy individuals for 24 h, and the protein expressions of collagen 1, collagen 3, IL-17RA and α-SMA were measured using Western blot. GAPDH was used as a loading control. The experiment was repeated three times, and the data are presented as means ± standard deviation.

    Journal: Arthritis Research & Therapy

    Article Title: Interleukin-17A promotes functional activation of systemic sclerosis patient-derived dermal vascular smooth muscle cells by extracellular-regulated protein kinases signalling pathway

    doi: 10.1186/s13075-014-0512-2

    Figure Lengend Snippet: IL-17A induces proliferation, collagen synthesis and secretion of SSc patient-derived DVSMCs via ERK1/2 signing pathway. (A) DVSMCs were pre-treated with PD 98059 (10 μM/ml) for 2 h before incubation with IL-17A at 100 ng/ml for 24 h, 48 h, 72 h, and cell proliferation was tested using cell counting kit-8. (B) The cells were pre-treated with PD98059 for 2 h before incubation with the serum of SSc patients or healthy individuals for 24 h, 48 h, 72 h, and cell proliferation was tested using CCK8. (C) The cells were pre-treated with PD 98059 for 2 h before incubation with IL-17A at 100 ng/ml for 24 h. The activity of collagen1α1 or collagen3α1 proximal promoter was detected using a dual luciferase reporter gene assay. (D) After being pre-treated with PD98059 for 2h before incubation with the serum of SSc patients or healthy individuals for 24 h, the activity of collagen1α1 or collagen3α1 proximal promoter was also detected. Data were represented as mean ratios of Firefly to Renilla luciferase activity. (E, F) The cells were pre-treated with PD 98059 for 2 h before incubation with IL-17A or SSc serum for 24 h, the gene expression of collagen 1 and collagen 3 was measured using real-time RT-PCR analysis. (G, H) The cells were pre-treated with PD 98059 for 2 h before incubation with IL-17A or SSc serum for 24 h, the concentration of collagen 1, collagen 3 was detected using ELISA. (I, J) The cells were pre-treated with PD98059 for 2 h before incubation with IL-17A, serum from SSc patients and healthy individuals for 24 h, and the protein expressions of collagen 1, collagen 3, IL-17RA and α-SMA were measured using Western blot. GAPDH was used as a loading control. The experiment was repeated three times, and the data are presented as means ± standard deviation.

    Article Snippet: After being blocked with 5% milk for 2 h at room temperature, the membranes were incubated using primary antibodies, including rabbit anti-human collagen 1 (1:1000, Abcam), rabbit anti-human collagen 3 (1:3000, Abcam), rabbit anti-human α-SMA (1:600, Abcam), goat anti-human IL-17RA (1:1000, Abcam), rabbit anti-human p38 (1:1000, Cell Signaling Technology, Boston, MA, USA), rabbit anti-human phospho-p38 MAPK (1:1000, Cell Signaling Technology), rabbit anti-human JNK (1:1000, Cell Signaling Technology), rabbit anti-human phospho-JNK (1:1000, Cell Signaling Technology), rabbit anti-human ERK1/2 (1:1000, Cell Signaling Technology), rabbit anti-human phospho-ERK1/2 (1:1000, Cell Signaling Technology), and rabbit anti-human GAPDH (1:1000, Cell Signaling Technology) overnight at 4°C.

    Techniques: Derivative Assay, Incubation, Cell Counting, Activity Assay, Luciferase, Reporter Gene Assay, Expressing, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Standard Deviation

    IL-17A derived from SSc patient serum promotes the proliferation, collagen synthesis and secretion of SSc patient-derived DVSMCs. (A) DVSMCs were treated with different doses of IL-17A for 24 h, 48 h, 72 h, and cell proliferation was tested using cell counting kit-8. (B) The cells were treated with the serum of SSc patients for 24 h, 48 h, 72 h, and cell proliferation was tested using CCK8. (C) The activity of collagen1α1 or collagen3α1 proximal promoter was test using a dual luciferase reporter gene assay after being treated with different doses of IL-17A for 24 h. (D) The activity of collagen1α1 or collagen3α1 proximal promoter was also detected after being treated with the serum of SSc patients and healthy subject for 24 h. Data were represented as mean ratios of Firefly to Renilla luciferase activity. (E) The cells were cultured in the indicated doses of IL-17A for 24 h, the gene expression of collagen 1 and collagen 3 was measured using real-time RT-PCR analysis. (F) The cells were stimulated with the serum of SSc patients and healthy subjects for 24 h, and the gene expression of collagen 1 and collagen 3 was measured using real-time RT-PCR analysis. (G) The cells were treated with different doses of IL-17A for 24 h, the concentration of collagen 1, collagen 3 was detected using ELISA. (H) The cells were treated with the serum of SSc patients and healthy subjects for 24 h, and the concentration of collagen 1, collagen 3 was also detected by ELISA. (I, J) The cells were treated with different doses of IL-17A, serum of SSc patients or healthy individuals for 24 h, and the expressions of collagen 1, collagen 3, IL-17RA and α-SMA were measured using Western blot. GAPDH was used as a loading control. The experiment was repeated three times, and the data are presented as means ± standard deviation.

    Journal: Arthritis Research & Therapy

    Article Title: Interleukin-17A promotes functional activation of systemic sclerosis patient-derived dermal vascular smooth muscle cells by extracellular-regulated protein kinases signalling pathway

    doi: 10.1186/s13075-014-0512-2

    Figure Lengend Snippet: IL-17A derived from SSc patient serum promotes the proliferation, collagen synthesis and secretion of SSc patient-derived DVSMCs. (A) DVSMCs were treated with different doses of IL-17A for 24 h, 48 h, 72 h, and cell proliferation was tested using cell counting kit-8. (B) The cells were treated with the serum of SSc patients for 24 h, 48 h, 72 h, and cell proliferation was tested using CCK8. (C) The activity of collagen1α1 or collagen3α1 proximal promoter was test using a dual luciferase reporter gene assay after being treated with different doses of IL-17A for 24 h. (D) The activity of collagen1α1 or collagen3α1 proximal promoter was also detected after being treated with the serum of SSc patients and healthy subject for 24 h. Data were represented as mean ratios of Firefly to Renilla luciferase activity. (E) The cells were cultured in the indicated doses of IL-17A for 24 h, the gene expression of collagen 1 and collagen 3 was measured using real-time RT-PCR analysis. (F) The cells were stimulated with the serum of SSc patients and healthy subjects for 24 h, and the gene expression of collagen 1 and collagen 3 was measured using real-time RT-PCR analysis. (G) The cells were treated with different doses of IL-17A for 24 h, the concentration of collagen 1, collagen 3 was detected using ELISA. (H) The cells were treated with the serum of SSc patients and healthy subjects for 24 h, and the concentration of collagen 1, collagen 3 was also detected by ELISA. (I, J) The cells were treated with different doses of IL-17A, serum of SSc patients or healthy individuals for 24 h, and the expressions of collagen 1, collagen 3, IL-17RA and α-SMA were measured using Western blot. GAPDH was used as a loading control. The experiment was repeated three times, and the data are presented as means ± standard deviation.

    Article Snippet: After being blocked with 5% milk for 2 h at room temperature, the membranes were incubated using primary antibodies, including rabbit anti-human collagen 1 (1:1000, Abcam), rabbit anti-human collagen 3 (1:3000, Abcam), rabbit anti-human α-SMA (1:600, Abcam), goat anti-human IL-17RA (1:1000, Abcam), rabbit anti-human p38 (1:1000, Cell Signaling Technology, Boston, MA, USA), rabbit anti-human phospho-p38 MAPK (1:1000, Cell Signaling Technology), rabbit anti-human JNK (1:1000, Cell Signaling Technology), rabbit anti-human phospho-JNK (1:1000, Cell Signaling Technology), rabbit anti-human ERK1/2 (1:1000, Cell Signaling Technology), rabbit anti-human phospho-ERK1/2 (1:1000, Cell Signaling Technology), and rabbit anti-human GAPDH (1:1000, Cell Signaling Technology) overnight at 4°C.

    Techniques: Derivative Assay, Cell Counting, Activity Assay, Luciferase, Reporter Gene Assay, Cell Culture, Expressing, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Standard Deviation

    Inhibition of TGF-β2 overcomes the downregulation of bone formation caused by LPS. a , b On day 28, alizarin red S solution was used to stain calcium deposits in cultures treated with TGF-β2 inhibitor and LPS. The dissolved mineral content of the medium decreased after LPS treatment. However, treatment with 1 μg · mL −1 TGF-β2 inhibitor neutralised the TGF-β2 secreted by LPS treatment. Interestingly, inhibition of TGF-β2 increased the osteogenic differentiation of DFSCs. c The results of ALPase activity measurements also supported the conclusion that inhibition of TGF-β2 increased the early stage of osteogenesis in DFSCs. d When the TGF-β2 secreted during LPS-induced inflammation was neutralised, the levels of the pro-inflammatory cytokines IL-6 and IL-8 decreased. In contrast, osteocalcin (OCN) and type 1 collagen (Col1) expression increased after treatment of the cells with TGF-β2 inhibitor during LPS-induced inflammation. e Treatment with 100 ng · mL −1 LPS for 30 min activated smad2/3 signalling. f DFSCs activated with LPS for 30 min were treated with 0.5 μg · mL −1 TGF-β2 inhibitor for 7 days during osteogenic differentiation. In the presence of a TGF-β2 inhibitor, Runx2 expression was overcome. The data are presented as the mean ± SD. * P

    Journal: International Journal of Oral Science

    Article Title: TGF-β2 downregulates osteogenesis under inflammatory conditions in dental follicle stem cells

    doi: 10.1038/s41368-018-0028-8

    Figure Lengend Snippet: Inhibition of TGF-β2 overcomes the downregulation of bone formation caused by LPS. a , b On day 28, alizarin red S solution was used to stain calcium deposits in cultures treated with TGF-β2 inhibitor and LPS. The dissolved mineral content of the medium decreased after LPS treatment. However, treatment with 1 μg · mL −1 TGF-β2 inhibitor neutralised the TGF-β2 secreted by LPS treatment. Interestingly, inhibition of TGF-β2 increased the osteogenic differentiation of DFSCs. c The results of ALPase activity measurements also supported the conclusion that inhibition of TGF-β2 increased the early stage of osteogenesis in DFSCs. d When the TGF-β2 secreted during LPS-induced inflammation was neutralised, the levels of the pro-inflammatory cytokines IL-6 and IL-8 decreased. In contrast, osteocalcin (OCN) and type 1 collagen (Col1) expression increased after treatment of the cells with TGF-β2 inhibitor during LPS-induced inflammation. e Treatment with 100 ng · mL −1 LPS for 30 min activated smad2/3 signalling. f DFSCs activated with LPS for 30 min were treated with 0.5 μg · mL −1 TGF-β2 inhibitor for 7 days during osteogenic differentiation. In the presence of a TGF-β2 inhibitor, Runx2 expression was overcome. The data are presented as the mean ± SD. * P

    Article Snippet: For immunohistochemical (IHC) staining, mouse anti-human mitochondria antibody (1:200, Chemicon, Temecula, CA), mouse anti-human osteocalcin antibody (1:100, Abcam, Cambridge, UK) and rabbit anti-human collagen type 1 (1:800, Abcam) were used followed by detection with an anti-mouse/rabbit HRP/DAB detection kit (Abcam).

    Techniques: Inhibition, Staining, Activity Assay, Expressing

    In vivo transplantation of normal and inflamed DFSCs. a H E staining was performed to determine the amount of hard tissue formed by transplanted normal and inflamed DFSCs with HA/TCP after 8 weeks transplantation to the dorsal skin of nude mice. Inflamed DFSCs showed significantly less hard tissue formation. b Dense collections of human mitochondria were detected in areas of hard tissue formation. c , d Hard tissue regions were positively stained for human osteocalcin and type 1 collagen to confirm hard tissue formation by transplanted normal and inflamed DFSCs. The two groups showed significantly different amounts of bone formation

    Journal: International Journal of Oral Science

    Article Title: TGF-β2 downregulates osteogenesis under inflammatory conditions in dental follicle stem cells

    doi: 10.1038/s41368-018-0028-8

    Figure Lengend Snippet: In vivo transplantation of normal and inflamed DFSCs. a H E staining was performed to determine the amount of hard tissue formed by transplanted normal and inflamed DFSCs with HA/TCP after 8 weeks transplantation to the dorsal skin of nude mice. Inflamed DFSCs showed significantly less hard tissue formation. b Dense collections of human mitochondria were detected in areas of hard tissue formation. c , d Hard tissue regions were positively stained for human osteocalcin and type 1 collagen to confirm hard tissue formation by transplanted normal and inflamed DFSCs. The two groups showed significantly different amounts of bone formation

    Article Snippet: For immunohistochemical (IHC) staining, mouse anti-human mitochondria antibody (1:200, Chemicon, Temecula, CA), mouse anti-human osteocalcin antibody (1:100, Abcam, Cambridge, UK) and rabbit anti-human collagen type 1 (1:800, Abcam) were used followed by detection with an anti-mouse/rabbit HRP/DAB detection kit (Abcam).

    Techniques: In Vivo, Transplantation Assay, Staining, Mouse Assay