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  • 99
    Abcam anti mcherry antibody
    CSCC-secreted exosomal miR-221-3p promotes lymphangiogenesis and lymphatic metastasis in vivo. a popliteal lymph node metastasis model was established in nude mice by inoculating the footpad with Siha/anti-221-3p (5 × 10 6 ) stably expressing <t>mCherry.</t> When footpad tumor size reached 50 mm 3 , exosomes (10 µg) secreted by Ect1/miR-NC, Ect1/miR-221-3p, Siha/anti-NC, or Siha/anti-221-3p were then injected into the center of the tumors ( n = 3/group, repeated twice) twice a week. After five injections, primary tumors reached a comparable size of ~ 150 mm 3 , and then footpad tumors and popliteal LNs were collected for study. a Staining of miR-221-3p and LYVE1 in serial sections of mice footpad tumors. Representative micrographs of positive staining are shown (left). The tumor cells are indicated by black arrows. The lymphatic vessels are indicated by red arrows. The correlation between miR-221-3p levels and PLVD was statistically analyzed (right). Scale bar, 20 µm. b Staining of mCherry in popliteal LNs from mice treated with the indicated exosomes. Representative micrographs are shown. Metastasis-positive LNs were identified by staining for cancer cell-expressed mCherry. Scale bar, upper panel, 200 µm; lower panel, 20 µm. c The ratio of metastasis-positive to total dissected popliteal LNs from mice treated with the indicated exosomes. ***, P
    Anti Mcherry Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti mcherry antibody/product/Abcam
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti mcherry antibody - by Bioz Stars, 2021-05
    99/100 stars
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    99
    Abcam mcherry
    CSCC-secreted exosomal miR-221-3p promotes lymphangiogenesis and lymphatic metastasis in vivo. a popliteal lymph node metastasis model was established in nude mice by inoculating the footpad with Siha/anti-221-3p (5 × 10 6 ) stably expressing <t>mCherry.</t> When footpad tumor size reached 50 mm 3 , exosomes (10 µg) secreted by Ect1/miR-NC, Ect1/miR-221-3p, Siha/anti-NC, or Siha/anti-221-3p were then injected into the center of the tumors ( n = 3/group, repeated twice) twice a week. After five injections, primary tumors reached a comparable size of ~ 150 mm 3 , and then footpad tumors and popliteal LNs were collected for study. a Staining of miR-221-3p and LYVE1 in serial sections of mice footpad tumors. Representative micrographs of positive staining are shown (left). The tumor cells are indicated by black arrows. The lymphatic vessels are indicated by red arrows. The correlation between miR-221-3p levels and PLVD was statistically analyzed (right). Scale bar, 20 µm. b Staining of mCherry in popliteal LNs from mice treated with the indicated exosomes. Representative micrographs are shown. Metastasis-positive LNs were identified by staining for cancer cell-expressed mCherry. Scale bar, upper panel, 200 µm; lower panel, 20 µm. c The ratio of metastasis-positive to total dissected popliteal LNs from mice treated with the indicated exosomes. ***, P
    Mcherry, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mcherry/product/Abcam
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mcherry - by Bioz Stars, 2021-05
    99/100 stars
      Buy from Supplier

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    Mouse Anti mCherry Polyclonal
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    Mouse Anti mCherry Polyclonal
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    mCherry Antibody is a Mouse Monoclonal against mCherry
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    N/A
    Rabbit polyclonal antibody to mCherry
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    CSCC-secreted exosomal miR-221-3p promotes lymphangiogenesis and lymphatic metastasis in vivo. a popliteal lymph node metastasis model was established in nude mice by inoculating the footpad with Siha/anti-221-3p (5 × 10 6 ) stably expressing mCherry. When footpad tumor size reached 50 mm 3 , exosomes (10 µg) secreted by Ect1/miR-NC, Ect1/miR-221-3p, Siha/anti-NC, or Siha/anti-221-3p were then injected into the center of the tumors ( n = 3/group, repeated twice) twice a week. After five injections, primary tumors reached a comparable size of ~ 150 mm 3 , and then footpad tumors and popliteal LNs were collected for study. a Staining of miR-221-3p and LYVE1 in serial sections of mice footpad tumors. Representative micrographs of positive staining are shown (left). The tumor cells are indicated by black arrows. The lymphatic vessels are indicated by red arrows. The correlation between miR-221-3p levels and PLVD was statistically analyzed (right). Scale bar, 20 µm. b Staining of mCherry in popliteal LNs from mice treated with the indicated exosomes. Representative micrographs are shown. Metastasis-positive LNs were identified by staining for cancer cell-expressed mCherry. Scale bar, upper panel, 200 µm; lower panel, 20 µm. c The ratio of metastasis-positive to total dissected popliteal LNs from mice treated with the indicated exosomes. ***, P

    Journal: Oncogene

    Article Title: Cervical squamous cell carcinoma-secreted exosomal miR-221-3p promotes lymphangiogenesis and lymphatic metastasis by targeting VASH1

    doi: 10.1038/s41388-018-0511-x

    Figure Lengend Snippet: CSCC-secreted exosomal miR-221-3p promotes lymphangiogenesis and lymphatic metastasis in vivo. a popliteal lymph node metastasis model was established in nude mice by inoculating the footpad with Siha/anti-221-3p (5 × 10 6 ) stably expressing mCherry. When footpad tumor size reached 50 mm 3 , exosomes (10 µg) secreted by Ect1/miR-NC, Ect1/miR-221-3p, Siha/anti-NC, or Siha/anti-221-3p were then injected into the center of the tumors ( n = 3/group, repeated twice) twice a week. After five injections, primary tumors reached a comparable size of ~ 150 mm 3 , and then footpad tumors and popliteal LNs were collected for study. a Staining of miR-221-3p and LYVE1 in serial sections of mice footpad tumors. Representative micrographs of positive staining are shown (left). The tumor cells are indicated by black arrows. The lymphatic vessels are indicated by red arrows. The correlation between miR-221-3p levels and PLVD was statistically analyzed (right). Scale bar, 20 µm. b Staining of mCherry in popliteal LNs from mice treated with the indicated exosomes. Representative micrographs are shown. Metastasis-positive LNs were identified by staining for cancer cell-expressed mCherry. Scale bar, upper panel, 200 µm; lower panel, 20 µm. c The ratio of metastasis-positive to total dissected popliteal LNs from mice treated with the indicated exosomes. ***, P

    Article Snippet: The primary antibodies were as follows: anti-VASH1 (ab176114, Abcam), anti-CD31 (ab28364, Abcam), anti-LYVE1 (ab33682, Abcam), and anti-mCherry antibody (ab167453, Abcam).

    Techniques: In Vivo, Mouse Assay, Stable Transfection, Expressing, Injection, Staining

    Spinal cord dynorphin neurons modulate itch and are downstream of the site of action of somatostatin. A, schematic diagram of the viral-based strategy employed to chemogenetically activate spinal cord neurons expressing dynorphin or nNOS. B, sagittal sections stained for mCherry showing that intraspinal injection of the AAV in Pdyn Cre and nNOS Cre mice produced expression with the expected distribution in spinal cord segments L3-L5, which innervate the hind-limb including the calf. Scale = 200 μm. C, Chemogenetic activation in the Pdyn Cre mouse. A transverse section of spinal cord taken from a Pdyn Cre mouse that had been injected with AAV2-flex-hM3Dq-mCherry and treated with CNO 2 hours prior to perfusion fixation. The section was immunostained to reveal mCherry (mCh, red), the somatostatin receptor Sst 2a (blue), Pax2 (gray) and Fos (green). Asterisks (*) show the cell bodies of 3 neurons that express hM3Dq-mCherry, Sst 2a receptor, Pax2 and Fos, indicating chemogenetic activation of inhibitory (Sst 2a for numbers). Arrowhead points to a Pax2 + (inhibitory) Sst 2a -expressing neuron that lacks mCherry and this cell was not Fos-positive. Scale = 10 μm. D, Chemogenetic activation in the nNOS CreERT2 mouse. Transverse section of spinal cord taken from a nNOS CreERT2 mouse injected with AAV2-flex-hM3Dq-mCherry and treated with CNO 2 hours prior to perfusion fixation. The section was immunostained to reveal mCherry (red), Sst 2a (blue), nNOS (gray) and Fos (green). Five cells showing varying levels of nNOS-immunoreactivity are visible. Two of these (asterisks) are stained for mCherry and Sst 2a (inhibitory nNOS cells) and these are Fos-positive. Of the 3 cells with weak nNOS-immunoreactivity, one (arrow) is positive for mCherry and Fos, but lacks Sst 2a , and is therefore likely an excitatory interneuron. The other two are not labelled with either mCherry or Fos: one of these is an Sst 2a -positive inhibitory neuron (single arrowhead), while the other lacks Sst 2a and is therefore likely to be an excitatory neuron (double arrowhead). This shows chemogenetic activation of nNOS cells, including inhibitory (Sst 2a for numbers). Scale = 20 μm. E. The time spent biting the calf in response to intradermal injection of chloroquine (100 μg) was reduced following chemogenetic activation (CNO) in Pdyn Cre mice, but there was no effect on itch responses in nNOS CreERT2 animals. Significant differences were assessed using two-sided unpaired Student t-tests (t 21 = 2.92, *p = 0.0082; and t 23 = 0.875, p = 0.391 ns not significant). Data represent means ± SEM (n=11, 12, 12, and 13 animals, for Pdyn Cre mice treated with CNO and vehicle and for nNOS CreERT2 mice treated with CNO and vehicle, respectively). mCherry-labelled injection sites (as shown in B) were verified in all of these experiments. F, DREADDq activation following intrathecal injection of AAV2-flex-hM3Dq significantly reduced numbers of itch bouts in Pdyn Cre mice injected into the nape of the neck with histamine (100 µg) and chloroquine (100 µg), and also when octreotide (100 ng) was administered intrathecally. Significant differences were assessed using two-sided unpaired Student t-tests (t 10 = 3.017, 3.053, 4.861, *p = 0.013, 0.0122, and 0.0007). Data represent means ± SEM (n= 6 animals).

    Journal: Nature neuroscience

    Article Title: Circuit dissection of the role of somatostatin in itch and pain

    doi: 10.1038/s41593-018-0119-z

    Figure Lengend Snippet: Spinal cord dynorphin neurons modulate itch and are downstream of the site of action of somatostatin. A, schematic diagram of the viral-based strategy employed to chemogenetically activate spinal cord neurons expressing dynorphin or nNOS. B, sagittal sections stained for mCherry showing that intraspinal injection of the AAV in Pdyn Cre and nNOS Cre mice produced expression with the expected distribution in spinal cord segments L3-L5, which innervate the hind-limb including the calf. Scale = 200 μm. C, Chemogenetic activation in the Pdyn Cre mouse. A transverse section of spinal cord taken from a Pdyn Cre mouse that had been injected with AAV2-flex-hM3Dq-mCherry and treated with CNO 2 hours prior to perfusion fixation. The section was immunostained to reveal mCherry (mCh, red), the somatostatin receptor Sst 2a (blue), Pax2 (gray) and Fos (green). Asterisks (*) show the cell bodies of 3 neurons that express hM3Dq-mCherry, Sst 2a receptor, Pax2 and Fos, indicating chemogenetic activation of inhibitory (Sst 2a for numbers). Arrowhead points to a Pax2 + (inhibitory) Sst 2a -expressing neuron that lacks mCherry and this cell was not Fos-positive. Scale = 10 μm. D, Chemogenetic activation in the nNOS CreERT2 mouse. Transverse section of spinal cord taken from a nNOS CreERT2 mouse injected with AAV2-flex-hM3Dq-mCherry and treated with CNO 2 hours prior to perfusion fixation. The section was immunostained to reveal mCherry (red), Sst 2a (blue), nNOS (gray) and Fos (green). Five cells showing varying levels of nNOS-immunoreactivity are visible. Two of these (asterisks) are stained for mCherry and Sst 2a (inhibitory nNOS cells) and these are Fos-positive. Of the 3 cells with weak nNOS-immunoreactivity, one (arrow) is positive for mCherry and Fos, but lacks Sst 2a , and is therefore likely an excitatory interneuron. The other two are not labelled with either mCherry or Fos: one of these is an Sst 2a -positive inhibitory neuron (single arrowhead), while the other lacks Sst 2a and is therefore likely to be an excitatory neuron (double arrowhead). This shows chemogenetic activation of nNOS cells, including inhibitory (Sst 2a for numbers). Scale = 20 μm. E. The time spent biting the calf in response to intradermal injection of chloroquine (100 μg) was reduced following chemogenetic activation (CNO) in Pdyn Cre mice, but there was no effect on itch responses in nNOS CreERT2 animals. Significant differences were assessed using two-sided unpaired Student t-tests (t 21 = 2.92, *p = 0.0082; and t 23 = 0.875, p = 0.391 ns not significant). Data represent means ± SEM (n=11, 12, 12, and 13 animals, for Pdyn Cre mice treated with CNO and vehicle and for nNOS CreERT2 mice treated with CNO and vehicle, respectively). mCherry-labelled injection sites (as shown in B) were verified in all of these experiments. F, DREADDq activation following intrathecal injection of AAV2-flex-hM3Dq significantly reduced numbers of itch bouts in Pdyn Cre mice injected into the nape of the neck with histamine (100 µg) and chloroquine (100 µg), and also when octreotide (100 ng) was administered intrathecally. Significant differences were assessed using two-sided unpaired Student t-tests (t 10 = 3.017, 3.053, 4.861, *p = 0.013, 0.0122, and 0.0007). Data represent means ± SEM (n= 6 animals).

    Article Snippet: The sections were incubated in anti-mCherry (rabbit antibody, Abcam, ab167453, 1:2000) for 3 days at 4°C and this was revealed with fluorescent-labelled species-specific secondary antibodies (Jackson Immunoresearch, West Grove, PA, USA).

    Techniques: Expressing, Staining, Injection, Mouse Assay, Produced, Activation Assay

    Stem cell potential and molecular characteristics of PDX1+ A undiff . a Isolation of PDX1+ and PDX1− A undiff from Plzf-mC/CreER; Pdx1 GFP/+ adults by flow cytometry. A undiff are mCherry+ CD9+ c-KIT−. Gates for GFP+ and GFP− cells were set according to Plzf-mC/CreER control (left profile). Percentage of cells in GFP+ gate from representative sample is shown ( n = 7). b Pdx1-GFP+ and GFP− adult A undiff fractions were transplanted into recipient testis and analysed 8 weeks later by whole-mount IF. Images show GFP and mCherry expression in representative donor colonies. Panels on right show higher magnification details of indicated areas and grayscale panels show individual immunostaining. Scale bar, 100 μm. c Colony-forming efficiency of Pdx1-GFP+ and GFP− A undiff fractions in transplantation assays from b . Data is presented as mean number of colonies per 10 5 donor cells ± s.e.m. ( n = 15 recipient testes for Pdx1-GFP− cells and n = 13 for Pdx1-GFP+ cells). Donor cells were pooled from a total of 7 Plzf-mC/CreER; Pdx1 GFP/+ adults. d Mean length ± s.e.m. of donor colonies was measured from tiled microscope images from experiment of c . e Mean number of GFP+ cells/donor colony ± s.e.m. were calculated for a set of whole-mount samples from transplant assay of c ( n = 58 colonies from Pdx1-GFP− cells and n = 63 from Pdx1-GFP+). f Heatmap illustrates expression of indicated genes from RNA-Seq analysis of Pdx1-GFP+ and GFP− A undiff fractions isolated as in a ( n = 4 mice). Differentially expressed genes (DEG) are in bold. Cut-off for DEG is false discovery rate (FDR)

    Journal: Nature Communications

    Article Title: Identification of dynamic undifferentiated cell states within the male germline

    doi: 10.1038/s41467-018-04827-z

    Figure Lengend Snippet: Stem cell potential and molecular characteristics of PDX1+ A undiff . a Isolation of PDX1+ and PDX1− A undiff from Plzf-mC/CreER; Pdx1 GFP/+ adults by flow cytometry. A undiff are mCherry+ CD9+ c-KIT−. Gates for GFP+ and GFP− cells were set according to Plzf-mC/CreER control (left profile). Percentage of cells in GFP+ gate from representative sample is shown ( n = 7). b Pdx1-GFP+ and GFP− adult A undiff fractions were transplanted into recipient testis and analysed 8 weeks later by whole-mount IF. Images show GFP and mCherry expression in representative donor colonies. Panels on right show higher magnification details of indicated areas and grayscale panels show individual immunostaining. Scale bar, 100 μm. c Colony-forming efficiency of Pdx1-GFP+ and GFP− A undiff fractions in transplantation assays from b . Data is presented as mean number of colonies per 10 5 donor cells ± s.e.m. ( n = 15 recipient testes for Pdx1-GFP− cells and n = 13 for Pdx1-GFP+ cells). Donor cells were pooled from a total of 7 Plzf-mC/CreER; Pdx1 GFP/+ adults. d Mean length ± s.e.m. of donor colonies was measured from tiled microscope images from experiment of c . e Mean number of GFP+ cells/donor colony ± s.e.m. were calculated for a set of whole-mount samples from transplant assay of c ( n = 58 colonies from Pdx1-GFP− cells and n = 63 from Pdx1-GFP+). f Heatmap illustrates expression of indicated genes from RNA-Seq analysis of Pdx1-GFP+ and GFP− A undiff fractions isolated as in a ( n = 4 mice). Differentially expressed genes (DEG) are in bold. Cut-off for DEG is false discovery rate (FDR)

    Article Snippet: Primary antibodies were as follows: Goat anti-GFRα1 (AF560, 1:250), anti-c-KIT (AF1356, 1:250), anti-mouse/rat PDX1 (AF2517, 1:250), anti-PLZF (AF2944, 1:500), anti-LIN28A (AF3757, 1:500) and anti-SOX3 (AF2569, 1:250) (R & D Systems), rabbit anti-mCherry (ab167453, 1:2000), anti-OCT4 (ab19857, 1:500), anti-SALL4 (ab29112, 1:2000) (Abcam), chicken anti-GFP (ab13970, 1:5000) and rat anti-germ cell-specific antigen (TRA98, 1:500) (Abcam), rat monoclonal anti-mCherry clone 16D7 (Thermo Fisher Scientific, 1:2000), rabbit monoclonal anti-Cyclin D1 clone SP4 (1:250) and mouse monoclonal anti-DNMT3A clone 64B1446 (1:200) (Novus Biologicals), rat anti-KI67 clone SolA15 (eBioscience, 1:250), rabbit monoclonal anti-c-KIT clone D13A2 (1:400) and anti-RARγ clone D3A4 (1:500) (Cell Signaling Technology) and rabbit monoclonal anti-mouse EOMES clone 1219A (R & D Systems, 1:1000).

    Techniques: Isolation, Flow Cytometry, Cytometry, Expressing, Immunostaining, Transplantation Assay, Microscopy, RNA Sequencing Assay, Mouse Assay

    Characterization of Plzf-mC/CreER transgenic mice. a , b Representative IF of adult Plzf-mC/CreER testis sections ( n = 3 mice). Tubule stages and populations are indicated. Scale bar, 50 μm. c Representative flow cytometry of fixed and permeabilized testis from Plzf-mC/CreER and wildtype (WT) adult testis ( n = 3 mice per genotype). PLZF+ cells are shown. d Plzf-mC/CreER; Z/EG mice injected daily with TAM for 5 days were harvested at indicated days after treatment. e Representative IF of testis sections from d ( n = 3 testes per time point). Insets show details of indicated areas. Scale bar, 50 μm. f Representative whole-mount IF from d . Inset shows detail of indicated area. Arrowheads: unlabelled GFRα1+ cells. Scale bar, 50 μm. g Flow cytometry of fixed and permeabilized testis cells from d . Graph indicates mean fraction of A undiff (PLZF+ c-KIT−) and PLZF+ c-KIT+ early differentiating cells expressing GFP ± standard error of mean (s.e.m.) ( n = 4 testes D3, D10 and D90, n = 6 testes D30). h Representative flow cytometry of live Plzf-mC/CreER testis cells. SSC is side scatter. mCherry+ gate was set according to WT. i Quantitative RT-PCR for spermatogonial markers from Plzf-mC/CreER cell fractions sorted as in h . mC− indicates mCherry−. Expression levels are corrected to β-actin and normalized so mean value of fraction #2 equals 1. Mean values ± s.e.m. are indicated ( n = 3 sorts, 2 mice pooled per sort). Significance vs. mCherry− cells is shown. j Violin plots of gene expression in 150 single cells of fraction #1 cells from h . Cells were gated according to Plzf and Vasa expression. k Left: mean in vitro colony-forming activity of Plzf-mC/CreER fractions ± s.e.m. isolated as in h ( n = 3 mice). mC− indicates mCherry−. Significance vs. mCherry− fraction is indicated. Right: representative IF of passaged cells from fraction #1 treated with vehicle or retinoic acid for 48 h ( n = 3). Scale bar, 50 μm. l Left: transplantation of cultured cells established from Plzf-mC/CreER fraction #1. Right: representative whole-mount IF of tubules 8 weeks post transplant demonstrating formation of mCherry+ colonies ( n = 5 recipients). Comparable spermatogenic capacity was observed upon transplantation of an independent line (3.90 colonies/10 5 cells; n = 4 recipient testes). Scale bar, 100 μm. Significance was calculated by two-tailed Student’s t -test (** P

    Journal: Nature Communications

    Article Title: Identification of dynamic undifferentiated cell states within the male germline

    doi: 10.1038/s41467-018-04827-z

    Figure Lengend Snippet: Characterization of Plzf-mC/CreER transgenic mice. a , b Representative IF of adult Plzf-mC/CreER testis sections ( n = 3 mice). Tubule stages and populations are indicated. Scale bar, 50 μm. c Representative flow cytometry of fixed and permeabilized testis from Plzf-mC/CreER and wildtype (WT) adult testis ( n = 3 mice per genotype). PLZF+ cells are shown. d Plzf-mC/CreER; Z/EG mice injected daily with TAM for 5 days were harvested at indicated days after treatment. e Representative IF of testis sections from d ( n = 3 testes per time point). Insets show details of indicated areas. Scale bar, 50 μm. f Representative whole-mount IF from d . Inset shows detail of indicated area. Arrowheads: unlabelled GFRα1+ cells. Scale bar, 50 μm. g Flow cytometry of fixed and permeabilized testis cells from d . Graph indicates mean fraction of A undiff (PLZF+ c-KIT−) and PLZF+ c-KIT+ early differentiating cells expressing GFP ± standard error of mean (s.e.m.) ( n = 4 testes D3, D10 and D90, n = 6 testes D30). h Representative flow cytometry of live Plzf-mC/CreER testis cells. SSC is side scatter. mCherry+ gate was set according to WT. i Quantitative RT-PCR for spermatogonial markers from Plzf-mC/CreER cell fractions sorted as in h . mC− indicates mCherry−. Expression levels are corrected to β-actin and normalized so mean value of fraction #2 equals 1. Mean values ± s.e.m. are indicated ( n = 3 sorts, 2 mice pooled per sort). Significance vs. mCherry− cells is shown. j Violin plots of gene expression in 150 single cells of fraction #1 cells from h . Cells were gated according to Plzf and Vasa expression. k Left: mean in vitro colony-forming activity of Plzf-mC/CreER fractions ± s.e.m. isolated as in h ( n = 3 mice). mC− indicates mCherry−. Significance vs. mCherry− fraction is indicated. Right: representative IF of passaged cells from fraction #1 treated with vehicle or retinoic acid for 48 h ( n = 3). Scale bar, 50 μm. l Left: transplantation of cultured cells established from Plzf-mC/CreER fraction #1. Right: representative whole-mount IF of tubules 8 weeks post transplant demonstrating formation of mCherry+ colonies ( n = 5 recipients). Comparable spermatogenic capacity was observed upon transplantation of an independent line (3.90 colonies/10 5 cells; n = 4 recipient testes). Scale bar, 100 μm. Significance was calculated by two-tailed Student’s t -test (** P

    Article Snippet: Primary antibodies were as follows: Goat anti-GFRα1 (AF560, 1:250), anti-c-KIT (AF1356, 1:250), anti-mouse/rat PDX1 (AF2517, 1:250), anti-PLZF (AF2944, 1:500), anti-LIN28A (AF3757, 1:500) and anti-SOX3 (AF2569, 1:250) (R & D Systems), rabbit anti-mCherry (ab167453, 1:2000), anti-OCT4 (ab19857, 1:500), anti-SALL4 (ab29112, 1:2000) (Abcam), chicken anti-GFP (ab13970, 1:5000) and rat anti-germ cell-specific antigen (TRA98, 1:500) (Abcam), rat monoclonal anti-mCherry clone 16D7 (Thermo Fisher Scientific, 1:2000), rabbit monoclonal anti-Cyclin D1 clone SP4 (1:250) and mouse monoclonal anti-DNMT3A clone 64B1446 (1:200) (Novus Biologicals), rat anti-KI67 clone SolA15 (eBioscience, 1:250), rabbit monoclonal anti-c-KIT clone D13A2 (1:400) and anti-RARγ clone D3A4 (1:500) (Cell Signaling Technology) and rabbit monoclonal anti-mouse EOMES clone 1219A (R & D Systems, 1:1000).

    Techniques: Transgenic Assay, Mouse Assay, Flow Cytometry, Cytometry, Injection, Expressing, Quantitative RT-PCR, In Vitro, Activity Assay, Isolation, Transplantation Assay, Cell Culture, Two Tailed Test

    Comparative analysis of reporter gene expression in spermatogonia. a , b Representative whole-mount IF of adult (8–10 weeks post natal) Plzf-mC/CreER; Sox2 GFP ( a ) and Plzf-mC/CreER; Oct4-GFP ( b ) seminiferous tubules. Inset panels show individual immunostaining within indicated area at higher magnification. Tubule staging and select A s and A pr are indicated. Scale bars, 50 μm. c Representative flow cytometry analysis of fixed and permeabilized testis cells from 1 of 3 Oct4-GFP and wild-type (WT) control adults. PLZF+ cell population is shown. Percentages of cells contained within gates are indicated. d Quantification of flow cytometry results from c . Graph indicates percentage of A undiff (PLZF+ c-KIT−) and cells initiating differentiation (PLZF+ c-KIT+) expressing GFP in Oct4-GFP adults. Horizontal bars indicate mean values ( n = 3 mice). e Graph shows percentage of GFRα1+ and SOX3+ spermatogonia positive for GFP in whole-mount seminiferous tubules of Oct4-GFP adults. Spermatogonial identity was confirmed by SALL4 counterstain. Horizontal bars indicate mean values ( n = 3 mice, > 200 cells scored per data point). f Representative whole-mount IF of adult Oct4-GFP seminiferous tubules for indicated markers ( n = 3 mice). Select A undiff cells are indicated. Scale bar, 50 μm. g Scheme summarizing expression patterns of indicated genes and transgenic reporters plus changes in cell morphology during spermatogonial differentiation. Markers used to isolate different spermatogonial populations are indicated. h Isolation of Oct4-GFP− and Oct4-GFP+ A undiff from Plzf-mC/CreER; Oct4-GFP adults by flow cytometry. Percentage of cells in each gate from a representative sample is indicated ( n = 6 mice). i Oct4-GFP− and GFP+ adult A undiff fractions were transplanted into recipients and analysed 8 weeks later by whole-mount IF. Images show GFP and mCherry expression in representative colonies. PLZF counterstain confirms A undiff and spermatogonial identity. Panels show higher magnification details of indicated areas. Scale bar, 100 μm. Graph shows colony-forming efficiency of Oct4-GFP+ and GFP− A undiff fractions. Data is presented as mean number of colonies per 10 5 donor cells ± s.e.m. ( n = 7 recipient testes for Oct4-GFP− cells and n = 6 for Oct4-GFP+ cells). Donor cells were pooled from 2 Plzf-mC/CreER; Oct4-GFP adults. Significance was calculated by two-tailed Student’s t -test (* P

    Journal: Nature Communications

    Article Title: Identification of dynamic undifferentiated cell states within the male germline

    doi: 10.1038/s41467-018-04827-z

    Figure Lengend Snippet: Comparative analysis of reporter gene expression in spermatogonia. a , b Representative whole-mount IF of adult (8–10 weeks post natal) Plzf-mC/CreER; Sox2 GFP ( a ) and Plzf-mC/CreER; Oct4-GFP ( b ) seminiferous tubules. Inset panels show individual immunostaining within indicated area at higher magnification. Tubule staging and select A s and A pr are indicated. Scale bars, 50 μm. c Representative flow cytometry analysis of fixed and permeabilized testis cells from 1 of 3 Oct4-GFP and wild-type (WT) control adults. PLZF+ cell population is shown. Percentages of cells contained within gates are indicated. d Quantification of flow cytometry results from c . Graph indicates percentage of A undiff (PLZF+ c-KIT−) and cells initiating differentiation (PLZF+ c-KIT+) expressing GFP in Oct4-GFP adults. Horizontal bars indicate mean values ( n = 3 mice). e Graph shows percentage of GFRα1+ and SOX3+ spermatogonia positive for GFP in whole-mount seminiferous tubules of Oct4-GFP adults. Spermatogonial identity was confirmed by SALL4 counterstain. Horizontal bars indicate mean values ( n = 3 mice, > 200 cells scored per data point). f Representative whole-mount IF of adult Oct4-GFP seminiferous tubules for indicated markers ( n = 3 mice). Select A undiff cells are indicated. Scale bar, 50 μm. g Scheme summarizing expression patterns of indicated genes and transgenic reporters plus changes in cell morphology during spermatogonial differentiation. Markers used to isolate different spermatogonial populations are indicated. h Isolation of Oct4-GFP− and Oct4-GFP+ A undiff from Plzf-mC/CreER; Oct4-GFP adults by flow cytometry. Percentage of cells in each gate from a representative sample is indicated ( n = 6 mice). i Oct4-GFP− and GFP+ adult A undiff fractions were transplanted into recipients and analysed 8 weeks later by whole-mount IF. Images show GFP and mCherry expression in representative colonies. PLZF counterstain confirms A undiff and spermatogonial identity. Panels show higher magnification details of indicated areas. Scale bar, 100 μm. Graph shows colony-forming efficiency of Oct4-GFP+ and GFP− A undiff fractions. Data is presented as mean number of colonies per 10 5 donor cells ± s.e.m. ( n = 7 recipient testes for Oct4-GFP− cells and n = 6 for Oct4-GFP+ cells). Donor cells were pooled from 2 Plzf-mC/CreER; Oct4-GFP adults. Significance was calculated by two-tailed Student’s t -test (* P

    Article Snippet: Primary antibodies were as follows: Goat anti-GFRα1 (AF560, 1:250), anti-c-KIT (AF1356, 1:250), anti-mouse/rat PDX1 (AF2517, 1:250), anti-PLZF (AF2944, 1:500), anti-LIN28A (AF3757, 1:500) and anti-SOX3 (AF2569, 1:250) (R & D Systems), rabbit anti-mCherry (ab167453, 1:2000), anti-OCT4 (ab19857, 1:500), anti-SALL4 (ab29112, 1:2000) (Abcam), chicken anti-GFP (ab13970, 1:5000) and rat anti-germ cell-specific antigen (TRA98, 1:500) (Abcam), rat monoclonal anti-mCherry clone 16D7 (Thermo Fisher Scientific, 1:2000), rabbit monoclonal anti-Cyclin D1 clone SP4 (1:250) and mouse monoclonal anti-DNMT3A clone 64B1446 (1:200) (Novus Biologicals), rat anti-KI67 clone SolA15 (eBioscience, 1:250), rabbit monoclonal anti-c-KIT clone D13A2 (1:400) and anti-RARγ clone D3A4 (1:500) (Cell Signaling Technology) and rabbit monoclonal anti-mouse EOMES clone 1219A (R & D Systems, 1:1000).

    Techniques: Expressing, Immunostaining, Flow Cytometry, Cytometry, Mouse Assay, Transgenic Assay, Isolation, Two Tailed Test

    Identification and characterization of distinct A undiff populations. a Oct4-GFP− and Oct4-GFP+ A undiff fractions were isolated from Plzf-mC/CreER; Oct4-GFP adults for gene expression profiling by microarray. A undiff fraction is mCherry+ CD9+ c-KIT−. b Confirmation of gene expression signatures of Oct4-GFP− and Oct4-GFP+ A undiff by quantitative RT-PCR. Candidate genes were selected from microarray analysis of a . Expression levels are corrected to those of β-actin and normalized so mean value of GFP− or GFP+ fractions equals 1. Mean values from 3-5 mice ± s.e.m. are indicated. Genes enriched in Oct4-GFP− and Oct4-GFP+ populations are shown in separate groups. Control genes Plzf and Vasa are shown. Significance was calculated by two-tailed Student’s t -test (* P

    Journal: Nature Communications

    Article Title: Identification of dynamic undifferentiated cell states within the male germline

    doi: 10.1038/s41467-018-04827-z

    Figure Lengend Snippet: Identification and characterization of distinct A undiff populations. a Oct4-GFP− and Oct4-GFP+ A undiff fractions were isolated from Plzf-mC/CreER; Oct4-GFP adults for gene expression profiling by microarray. A undiff fraction is mCherry+ CD9+ c-KIT−. b Confirmation of gene expression signatures of Oct4-GFP− and Oct4-GFP+ A undiff by quantitative RT-PCR. Candidate genes were selected from microarray analysis of a . Expression levels are corrected to those of β-actin and normalized so mean value of GFP− or GFP+ fractions equals 1. Mean values from 3-5 mice ± s.e.m. are indicated. Genes enriched in Oct4-GFP− and Oct4-GFP+ populations are shown in separate groups. Control genes Plzf and Vasa are shown. Significance was calculated by two-tailed Student’s t -test (* P

    Article Snippet: Primary antibodies were as follows: Goat anti-GFRα1 (AF560, 1:250), anti-c-KIT (AF1356, 1:250), anti-mouse/rat PDX1 (AF2517, 1:250), anti-PLZF (AF2944, 1:500), anti-LIN28A (AF3757, 1:500) and anti-SOX3 (AF2569, 1:250) (R & D Systems), rabbit anti-mCherry (ab167453, 1:2000), anti-OCT4 (ab19857, 1:500), anti-SALL4 (ab29112, 1:2000) (Abcam), chicken anti-GFP (ab13970, 1:5000) and rat anti-germ cell-specific antigen (TRA98, 1:500) (Abcam), rat monoclonal anti-mCherry clone 16D7 (Thermo Fisher Scientific, 1:2000), rabbit monoclonal anti-Cyclin D1 clone SP4 (1:250) and mouse monoclonal anti-DNMT3A clone 64B1446 (1:200) (Novus Biologicals), rat anti-KI67 clone SolA15 (eBioscience, 1:250), rabbit monoclonal anti-c-KIT clone D13A2 (1:400) and anti-RARγ clone D3A4 (1:500) (Cell Signaling Technology) and rabbit monoclonal anti-mouse EOMES clone 1219A (R & D Systems, 1:1000).

    Techniques: Isolation, Expressing, Microarray, Quantitative RT-PCR, Mouse Assay, Two Tailed Test