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igg2a vector mouse g2a ch domain amplification  (Vector Laboratories)


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    Vector Laboratories igg2a vector mouse g2a ch domain amplification
    Figure 1. Schematic representation of the R-mAb pipeline. (A) Schematic of cloning, expression and validation pipeline. Orange steps involve VH and VL regions of individual hybridomas, blue steps involve steps involving backbone components, and green step involves expression of target for R-mAb validation. (B) Schematic shows the separate elements of the R-mAb expression plasmid involved in coexpression of light (green) and heavy (blue) chains as driven by two CMV promoters (orange). Hybridoma-derived VL and VH domain PCR products are fused to a joining fragment comprising a k light chain constant domain (CL) and the k light chain polyA tail sequences (k pA), a CMV promoter for heavy chain expression, and an ER signal/leader sequence (L) for translocation of the heavy chain across the ER membrane. PCR-mediated fusion of these three elements is followed by their insertion into the p1316 plasmid that contains an upstream CMV promoter for light chain expression, and an ER signal/leader sequence (L) for translocation of the light chain across the ER membrane. Downstream of the insert is a heavy chain constant domain (CH) that is either g1 or <t>g2a</t> depending on the plasmid, followed by the SV40 polyA tail (SV40 pA). DOI: https://doi.org/10.7554/eLife.43322.003
    Igg2a Vector Mouse G2a Ch Domain Amplification, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 96/100, based on 7323 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/igg2a vector mouse g2a ch domain amplification/product/Vector Laboratories
    Average 96 stars, based on 7323 article reviews
    igg2a vector mouse g2a ch domain amplification - by Bioz Stars, 2026-03
    96/100 stars

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    1) Product Images from "A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain"

    Article Title: A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain

    Journal: eLife

    doi: 10.7554/elife.43322

    Figure 1. Schematic representation of the R-mAb pipeline. (A) Schematic of cloning, expression and validation pipeline. Orange steps involve VH and VL regions of individual hybridomas, blue steps involve steps involving backbone components, and green step involves expression of target for R-mAb validation. (B) Schematic shows the separate elements of the R-mAb expression plasmid involved in coexpression of light (green) and heavy (blue) chains as driven by two CMV promoters (orange). Hybridoma-derived VL and VH domain PCR products are fused to a joining fragment comprising a k light chain constant domain (CL) and the k light chain polyA tail sequences (k pA), a CMV promoter for heavy chain expression, and an ER signal/leader sequence (L) for translocation of the heavy chain across the ER membrane. PCR-mediated fusion of these three elements is followed by their insertion into the p1316 plasmid that contains an upstream CMV promoter for light chain expression, and an ER signal/leader sequence (L) for translocation of the light chain across the ER membrane. Downstream of the insert is a heavy chain constant domain (CH) that is either g1 or g2a depending on the plasmid, followed by the SV40 polyA tail (SV40 pA). DOI: https://doi.org/10.7554/eLife.43322.003
    Figure Legend Snippet: Figure 1. Schematic representation of the R-mAb pipeline. (A) Schematic of cloning, expression and validation pipeline. Orange steps involve VH and VL regions of individual hybridomas, blue steps involve steps involving backbone components, and green step involves expression of target for R-mAb validation. (B) Schematic shows the separate elements of the R-mAb expression plasmid involved in coexpression of light (green) and heavy (blue) chains as driven by two CMV promoters (orange). Hybridoma-derived VL and VH domain PCR products are fused to a joining fragment comprising a k light chain constant domain (CL) and the k light chain polyA tail sequences (k pA), a CMV promoter for heavy chain expression, and an ER signal/leader sequence (L) for translocation of the heavy chain across the ER membrane. PCR-mediated fusion of these three elements is followed by their insertion into the p1316 plasmid that contains an upstream CMV promoter for light chain expression, and an ER signal/leader sequence (L) for translocation of the light chain across the ER membrane. Downstream of the insert is a heavy chain constant domain (CH) that is either g1 or g2a depending on the plasmid, followed by the SV40 polyA tail (SV40 pA). DOI: https://doi.org/10.7554/eLife.43322.003

    Techniques Used: Cloning, Expressing, Biomarker Discovery, Plasmid Preparation, Derivative Assay, Sequencing, Translocation Assay, Membrane

    Figure 2. Cloning of VL and VH domain sequences from hybridomas into the R-mAb expression plasmid. (A) Agarose gel analysis of VL and VH domain PCR products amplified from cDNA synthesized from RNA extracted from the N59/36 (anti-NR2B/GRIN2B) and K39/25 (anti-Kv2.1/KCNB1) hybridomas. The expected size of mouse IgG VL and VH domains is » 360 bp. (B) Agarose gel analysis of VH and digested VL fragments joined by fusion PCR (F-PCR) to the P1316-derived joining fragment to create a dual IgG chain cassette. (C) Agarose gel analysis of colony PCR samples of transformants from the N59/36 R-mAb project. (D) Agarose gel analysis of products of restriction enzyme digestion of N59/36 plasmid DNA with NotI and AscI. The plasmid backbone is seven kbp, and the intact insert comprising the VL and VH domains and the intervening joining fragment is 2.4 kbp. (E) Agarose gel analysis of PCR products of VL domain cDNA synthesized from RNA extracted from mouse splenocytes, the fusion partner Sp2/0-Ag14, and various hybridomas after digestion with the BciVI restriction enzyme to cleave the Sp2/0-Ag14-derived aberrant light chain product. The intact VL domains are » 360 bp, and the digested aberrant light chains » 180 bp. DOI: https://doi.org/10.7554/eLife.43322.004
    Figure Legend Snippet: Figure 2. Cloning of VL and VH domain sequences from hybridomas into the R-mAb expression plasmid. (A) Agarose gel analysis of VL and VH domain PCR products amplified from cDNA synthesized from RNA extracted from the N59/36 (anti-NR2B/GRIN2B) and K39/25 (anti-Kv2.1/KCNB1) hybridomas. The expected size of mouse IgG VL and VH domains is » 360 bp. (B) Agarose gel analysis of VH and digested VL fragments joined by fusion PCR (F-PCR) to the P1316-derived joining fragment to create a dual IgG chain cassette. (C) Agarose gel analysis of colony PCR samples of transformants from the N59/36 R-mAb project. (D) Agarose gel analysis of products of restriction enzyme digestion of N59/36 plasmid DNA with NotI and AscI. The plasmid backbone is seven kbp, and the intact insert comprising the VL and VH domains and the intervening joining fragment is 2.4 kbp. (E) Agarose gel analysis of PCR products of VL domain cDNA synthesized from RNA extracted from mouse splenocytes, the fusion partner Sp2/0-Ag14, and various hybridomas after digestion with the BciVI restriction enzyme to cleave the Sp2/0-Ag14-derived aberrant light chain product. The intact VL domains are » 360 bp, and the digested aberrant light chains » 180 bp. DOI: https://doi.org/10.7554/eLife.43322.004

    Techniques Used: Cloning, Expressing, Plasmid Preparation, Agarose Gel Electrophoresis, Amplification, Synthesized, Derivative Assay

    Figure 3. Validation of subclass-switched anti-PSD-95 K28/43R R-mAb. (A) Validation of the K28/43R R-mAb in heterologous cells. COS-1 cells transiently transfected to express human PSD-95 in a subset of cells were immunolabeled with K28/43 mAb (IgG2a) alone (top row), K28/43R R-mAb (IgG1) alone (middle row), or K28/43 mAb plus K28/43R R-mAb (bottom row). Immunolabeling in all samples was detected with a cocktail of anti-mouse IgG2a (red, for the K28/43 mAb) and anti-mouse IgG1 (green, for the K28/43R R-mAb) subclass-specific Alexa Fluor conjugated secondary antibodies. Labeling in blue is for the DNA-specific dye Hoechst 33258 and shows nuclei of both transfected and untransfected cells. Scale bar in the lower right merged panel = 30 mm and holds for all panels in A. (B) Validation of the K28/43R R-mAb in brain sections. A brain section from an adult rat was immunolabeled with K28/43 mAb plus K28/43R R-mAb and immunolabeling detected with a cocktail of anti-mouse IgG2a (red, for K28/43 mAb) and anti-mouse IgG1 (green, for K28/43R R-mAb) subclass-specific Alexa Fluor conjugated secondary antibodies. Cell nuclei are labeled with the DNA- specific dye Hoechst 33258 (blue). The region of interest shown is from cerebellar cortex. Scale bar in the left panel = 100 mm, and in the right merged panel = 30 mm. (C) Immunoblots against brain membranes and COS cell lysates over-expressing various members of the MAGUK superfamily of scaffolding proteins. To confirm expression of MAGUK proteins, immunoblots were probed with rabbit anti-PSD-95 (red). K28/86 is an anti-MAGUK mAb. Primary antibodies were detected with the appropriate combinations of fluorescently labeled species-specific anti-rabbit and subclass-specific anti-mouse IgG secondary Abs as indicated. Control indicates COS cells transfected with an empty vector. DOI: https://doi.org/10.7554/eLife.43322.006
    Figure Legend Snippet: Figure 3. Validation of subclass-switched anti-PSD-95 K28/43R R-mAb. (A) Validation of the K28/43R R-mAb in heterologous cells. COS-1 cells transiently transfected to express human PSD-95 in a subset of cells were immunolabeled with K28/43 mAb (IgG2a) alone (top row), K28/43R R-mAb (IgG1) alone (middle row), or K28/43 mAb plus K28/43R R-mAb (bottom row). Immunolabeling in all samples was detected with a cocktail of anti-mouse IgG2a (red, for the K28/43 mAb) and anti-mouse IgG1 (green, for the K28/43R R-mAb) subclass-specific Alexa Fluor conjugated secondary antibodies. Labeling in blue is for the DNA-specific dye Hoechst 33258 and shows nuclei of both transfected and untransfected cells. Scale bar in the lower right merged panel = 30 mm and holds for all panels in A. (B) Validation of the K28/43R R-mAb in brain sections. A brain section from an adult rat was immunolabeled with K28/43 mAb plus K28/43R R-mAb and immunolabeling detected with a cocktail of anti-mouse IgG2a (red, for K28/43 mAb) and anti-mouse IgG1 (green, for K28/43R R-mAb) subclass-specific Alexa Fluor conjugated secondary antibodies. Cell nuclei are labeled with the DNA- specific dye Hoechst 33258 (blue). The region of interest shown is from cerebellar cortex. Scale bar in the left panel = 100 mm, and in the right merged panel = 30 mm. (C) Immunoblots against brain membranes and COS cell lysates over-expressing various members of the MAGUK superfamily of scaffolding proteins. To confirm expression of MAGUK proteins, immunoblots were probed with rabbit anti-PSD-95 (red). K28/86 is an anti-MAGUK mAb. Primary antibodies were detected with the appropriate combinations of fluorescently labeled species-specific anti-rabbit and subclass-specific anti-mouse IgG secondary Abs as indicated. Control indicates COS cells transfected with an empty vector. DOI: https://doi.org/10.7554/eLife.43322.006

    Techniques Used: Biomarker Discovery, Transfection, Immunolabeling, Labeling, Western Blot, Expressing, Scaffolding, Control, Plasmid Preparation

    Figure 4. Multiplex immunolabeling with subclass-switched recombinant antibodies in adult rat brain. (A) A section from neocortex labeled with anti- pan-Nav R-mAb K58/35R (IgG2a, red) at nodes of Ranvier and AIS (arrows), anti-CASPR mAb K65/35 (IgG1, green) at paranodes, and anti-Kv2.1 rabbit polyclonal (KC) antibody (blue) on somata and proximal dendrites. Scale bar = 150 mm. Insets (dashed box) show details of labeling for pan-Nav (red) and CASPR (green) at the node and paranodes (arrows), respectively, at a single node of Ranvier as indicated by box in main panel. (B) A section Figure 4 continued on next page
    Figure Legend Snippet: Figure 4. Multiplex immunolabeling with subclass-switched recombinant antibodies in adult rat brain. (A) A section from neocortex labeled with anti- pan-Nav R-mAb K58/35R (IgG2a, red) at nodes of Ranvier and AIS (arrows), anti-CASPR mAb K65/35 (IgG1, green) at paranodes, and anti-Kv2.1 rabbit polyclonal (KC) antibody (blue) on somata and proximal dendrites. Scale bar = 150 mm. Insets (dashed box) show details of labeling for pan-Nav (red) and CASPR (green) at the node and paranodes (arrows), respectively, at a single node of Ranvier as indicated by box in main panel. (B) A section Figure 4 continued on next page

    Techniques Used: Multiplex Assay, Immunolabeling, Recombinant, Labeling

    Figure 5. Cloning of anti-Kv2.1 D3/71 VL and VH domain cDNAs from a nonviable hybridoma. (A) Agarose gel analysis of PCR amplified VL and VH domains from cDNA synthesized from RNA extracted from the non-viable D3/71 hybridoma. The panel to the right shows the VL after digestion with the BciVI restriction enzyme to cleave the Sp2/0-Ag14-derived aberrant light chain product. The expected size of mouse IgG VL and VH domains is » 360 bp, and of the cleaved aberrant VL domain is » 180 bp. (B) Agarose gel analysis of D3/71 VH and digested VL fragments joined by fusion PCR (F- PCR) to the P1316 joining fragment to create a dual IgG chain cassette. (C) Agarose gel analysis of colony PCR samples of transformants from the of D3/71 R-mAb project. (D) Agarose gel analysis of products of restriction enzyme digestion of D3/71 plasmid DNA with NotI and AscI. The plasmid backbone is seven kbp, and the intact insert comprising the VL and VH domains and the intervening joining fragment is 2.4 kbp. DOI: https://doi.org/10.7554/eLife.43322.008
    Figure Legend Snippet: Figure 5. Cloning of anti-Kv2.1 D3/71 VL and VH domain cDNAs from a nonviable hybridoma. (A) Agarose gel analysis of PCR amplified VL and VH domains from cDNA synthesized from RNA extracted from the non-viable D3/71 hybridoma. The panel to the right shows the VL after digestion with the BciVI restriction enzyme to cleave the Sp2/0-Ag14-derived aberrant light chain product. The expected size of mouse IgG VL and VH domains is » 360 bp, and of the cleaved aberrant VL domain is » 180 bp. (B) Agarose gel analysis of D3/71 VH and digested VL fragments joined by fusion PCR (F- PCR) to the P1316 joining fragment to create a dual IgG chain cassette. (C) Agarose gel analysis of colony PCR samples of transformants from the of D3/71 R-mAb project. (D) Agarose gel analysis of products of restriction enzyme digestion of D3/71 plasmid DNA with NotI and AscI. The plasmid backbone is seven kbp, and the intact insert comprising the VL and VH domains and the intervening joining fragment is 2.4 kbp. DOI: https://doi.org/10.7554/eLife.43322.008

    Techniques Used: Cloning, Agarose Gel Electrophoresis, Amplification, Synthesized, Derivative Assay, Plasmid Preparation

    Figure 6. Recovery of a functional anti-Kv2.1 D3/71R R-mAb from nonviable hybridomas. (A) Validation of the D3/71R R-mAb in heterologous cells. COS-1 cells transiently transfected to express rat Kv2.1 in a subset of cells were immunolabeled with K89/34 mAb (IgG1) alone (top row), D3/71R R-mAb (IgG2a) alone (middle row), or K89/34 mAb plus D3/71R R-mAb (bottom row). Immunolabeling in all samples was detected with a cocktail of anti-mouse IgG1 (green, for the K89/34 mAb) and anti-mouse IgG2a (red, for the D3/71R R-mAb) subclass-specific Alexa Fluor conjugated secondary antibodies. (B) Validation of the subclass-switched K89/34R R-mAb in heterologous cells. COS-1 cells transiently transfected to express rat Kv2.1 in a subset of cells were immunolabeled with K89/34 mAb (IgG1) alone (top row), K89/34R R-mAb (IgG2a) alone (middle row), or K89/34 mAb plus K89/34R R-mAb (bottom row). Immunolabeling in all samples was detected with a cocktail of anti-mouse IgG1 (green, for the K89/34 mAb) and anti-mouse IgG2a (red, for the K89/34R R-mAb) subclass-specific Alexa Fluor conjugated secondary antibodies. Labeling in blue in panels A and B is for the DNA-specific dye Hoechst 33258 and shows nuclei of both transfected and untransfected cells. Scale bar in the lower right merged panel = 30 mm and holds for all panels in A and B. (C) Validation of the D3/71R R-mAb in brain sections. A brain section from an adult rat was immunolabeled with K89/34 mAb plus D3/71 R-mAb and the immunolabeling detected with a cocktail of anti-mouse IgG1 (green, for the K89/34 mAb) and anti-mouse IgG2a (red, for the D3/71R R-mAb) subclass-specific Alexa Fluor conjugated secondary antibodies. Cell nuclei are labeled with the DNA-specific dye Hoechst 33258 (blue). Region of interest shown is from neocortex. Scale bar = 30 mm. (D) Strip immunoblots on a crude rat brain membrane fraction immunolabeled with the K89/34 mAb, the K89/34R R-mAb, and the D3/71 R-mAb as indicated. Immunolabeling was detected on autoradiography film after treatment of strip immunoblots with HRP-conjugated anti-mouse IgG-specific secondary antibody and ECL. DOI: https://doi.org/10.7554/eLife.43322.009
    Figure Legend Snippet: Figure 6. Recovery of a functional anti-Kv2.1 D3/71R R-mAb from nonviable hybridomas. (A) Validation of the D3/71R R-mAb in heterologous cells. COS-1 cells transiently transfected to express rat Kv2.1 in a subset of cells were immunolabeled with K89/34 mAb (IgG1) alone (top row), D3/71R R-mAb (IgG2a) alone (middle row), or K89/34 mAb plus D3/71R R-mAb (bottom row). Immunolabeling in all samples was detected with a cocktail of anti-mouse IgG1 (green, for the K89/34 mAb) and anti-mouse IgG2a (red, for the D3/71R R-mAb) subclass-specific Alexa Fluor conjugated secondary antibodies. (B) Validation of the subclass-switched K89/34R R-mAb in heterologous cells. COS-1 cells transiently transfected to express rat Kv2.1 in a subset of cells were immunolabeled with K89/34 mAb (IgG1) alone (top row), K89/34R R-mAb (IgG2a) alone (middle row), or K89/34 mAb plus K89/34R R-mAb (bottom row). Immunolabeling in all samples was detected with a cocktail of anti-mouse IgG1 (green, for the K89/34 mAb) and anti-mouse IgG2a (red, for the K89/34R R-mAb) subclass-specific Alexa Fluor conjugated secondary antibodies. Labeling in blue in panels A and B is for the DNA-specific dye Hoechst 33258 and shows nuclei of both transfected and untransfected cells. Scale bar in the lower right merged panel = 30 mm and holds for all panels in A and B. (C) Validation of the D3/71R R-mAb in brain sections. A brain section from an adult rat was immunolabeled with K89/34 mAb plus D3/71 R-mAb and the immunolabeling detected with a cocktail of anti-mouse IgG1 (green, for the K89/34 mAb) and anti-mouse IgG2a (red, for the D3/71R R-mAb) subclass-specific Alexa Fluor conjugated secondary antibodies. Cell nuclei are labeled with the DNA-specific dye Hoechst 33258 (blue). Region of interest shown is from neocortex. Scale bar = 30 mm. (D) Strip immunoblots on a crude rat brain membrane fraction immunolabeled with the K89/34 mAb, the K89/34R R-mAb, and the D3/71 R-mAb as indicated. Immunolabeling was detected on autoradiography film after treatment of strip immunoblots with HRP-conjugated anti-mouse IgG-specific secondary antibody and ECL. DOI: https://doi.org/10.7554/eLife.43322.009

    Techniques Used: Functional Assay, Biomarker Discovery, Transfection, Immunolabeling, Labeling, Stripping Membranes, Western Blot, Membrane, Autoradiography



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    igg2a vector mouse g2a ch domain amplification  (Vector Laboratories)
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    Vector Laboratories igg2a vector mouse g2a ch domain amplification
    Figure 1. Schematic representation of the R-mAb pipeline. (A) Schematic of cloning, expression and validation pipeline. Orange steps involve VH and VL regions of individual hybridomas, blue steps involve steps involving backbone components, and green step involves expression of target for R-mAb validation. (B) Schematic shows the separate elements of the R-mAb expression plasmid involved in coexpression of light (green) and heavy (blue) chains as driven by two CMV promoters (orange). Hybridoma-derived VL and VH domain PCR products are fused to a joining fragment comprising a k light chain constant domain (CL) and the k light chain polyA tail sequences (k pA), a CMV promoter for heavy chain expression, and an ER signal/leader sequence (L) for translocation of the heavy chain across the ER membrane. PCR-mediated fusion of these three elements is followed by their insertion into the p1316 plasmid that contains an upstream CMV promoter for light chain expression, and an ER signal/leader sequence (L) for translocation of the light chain across the ER membrane. Downstream of the insert is a heavy chain constant domain (CH) that is either g1 or <t>g2a</t> depending on the plasmid, followed by the SV40 polyA tail (SV40 pA). DOI: https://doi.org/10.7554/eLife.43322.003
    Igg2a Vector Mouse G2a Ch Domain Amplification, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/igg2a vector mouse g2a ch domain amplification/product/Vector Laboratories
    Average 96 stars, based on 1 article reviews
    igg2a vector mouse g2a ch domain amplification - by Bioz Stars, 2026-03
    96/100 stars
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    Figure 1. Schematic representation of the R-mAb pipeline. (A) Schematic of cloning, expression and validation pipeline. Orange steps involve VH and VL regions of individual hybridomas, blue steps involve steps involving backbone components, and green step involves expression of target for R-mAb validation. (B) Schematic shows the separate elements of the R-mAb expression plasmid involved in coexpression of light (green) and heavy (blue) chains as driven by two CMV promoters (orange). Hybridoma-derived VL and VH domain PCR products are fused to a joining fragment comprising a k light chain constant domain (CL) and the k light chain polyA tail sequences (k pA), a CMV promoter for heavy chain expression, and an ER signal/leader sequence (L) for translocation of the heavy chain across the ER membrane. PCR-mediated fusion of these three elements is followed by their insertion into the p1316 plasmid that contains an upstream CMV promoter for light chain expression, and an ER signal/leader sequence (L) for translocation of the light chain across the ER membrane. Downstream of the insert is a heavy chain constant domain (CH) that is either g1 or g2a depending on the plasmid, followed by the SV40 polyA tail (SV40 pA). DOI: https://doi.org/10.7554/eLife.43322.003

    Journal: eLife

    Article Title: A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain

    doi: 10.7554/elife.43322

    Figure Lengend Snippet: Figure 1. Schematic representation of the R-mAb pipeline. (A) Schematic of cloning, expression and validation pipeline. Orange steps involve VH and VL regions of individual hybridomas, blue steps involve steps involving backbone components, and green step involves expression of target for R-mAb validation. (B) Schematic shows the separate elements of the R-mAb expression plasmid involved in coexpression of light (green) and heavy (blue) chains as driven by two CMV promoters (orange). Hybridoma-derived VL and VH domain PCR products are fused to a joining fragment comprising a k light chain constant domain (CL) and the k light chain polyA tail sequences (k pA), a CMV promoter for heavy chain expression, and an ER signal/leader sequence (L) for translocation of the heavy chain across the ER membrane. PCR-mediated fusion of these three elements is followed by their insertion into the p1316 plasmid that contains an upstream CMV promoter for light chain expression, and an ER signal/leader sequence (L) for translocation of the light chain across the ER membrane. Downstream of the insert is a heavy chain constant domain (CH) that is either g1 or g2a depending on the plasmid, followed by the SV40 polyA tail (SV40 pA). DOI: https://doi.org/10.7554/eLife.43322.003

    Article Snippet: Amplification of the Joining Fragment (Crosnier et al., 2010) Primer 21: 5’- GGGCTGATGCTGCACCAACTGTA-3’ Primer 26: 5’-ACTGCTTGAGGCTGGACTCGTGAACAATAGCAGC-3’ Colony PCR: UpNotI: 5’-TTTCAGACCCAGGTACTCAT-3’ DownAscI: 5’-GGGCAGCAGATCCAGGGGCC-3’ (reverse primer for IgG1 vector) Rev IgG2a: 5’- ACCCTTGACCAGGCATCCTAGAGT- 3’ (reverse primer for IgG2a vector) Mouse g2a CH domain amplification (restriction sites are underlined): IgG2a-F-AscI: 5’-ATATCACGGCGCGCCCAACAGCCCCATCGGTCTATCCA-3’ IgG2a-R-XbaI: 5’GACTGATCTAGATCATTTACCCGGAGTCCGGGAGAA-3’ R-mAb Sequencing: Forward strand of VL region: UpNotI = 5’-TTTCAGACCCAGGTACTCAT-3’ Reverse strand of VL region (IgG2a plasmids): Seq_VL_Rev_IgG2a = 5’ - CCAACTGTTCAG- GACGCCATT 3’ Forward strand of VH region: VH_seq_Forward = 5’- TCCCAGGCCACCATGAA 3’ Reverse strand of VH region (IgG1 plasmids): DownAscI = 5’-GGGCAGCAGATCCAGGGGCC-3’ Reverse strand of VH region (IgG2a plasmids): Rev IgG2a = 5’- ACCCTTGACCAGGCATCC TAGAGT- 3’ RNA preparation from cryopreserved hybridomas and RT-PCR The Ambion RNAqueous 96 Extraction kit (Thermo Fisher Cat# AM1920) was used for high throughput RNA extraction.

    Techniques: Cloning, Expressing, Biomarker Discovery, Plasmid Preparation, Derivative Assay, Sequencing, Translocation Assay, Membrane

    Figure 2. Cloning of VL and VH domain sequences from hybridomas into the R-mAb expression plasmid. (A) Agarose gel analysis of VL and VH domain PCR products amplified from cDNA synthesized from RNA extracted from the N59/36 (anti-NR2B/GRIN2B) and K39/25 (anti-Kv2.1/KCNB1) hybridomas. The expected size of mouse IgG VL and VH domains is » 360 bp. (B) Agarose gel analysis of VH and digested VL fragments joined by fusion PCR (F-PCR) to the P1316-derived joining fragment to create a dual IgG chain cassette. (C) Agarose gel analysis of colony PCR samples of transformants from the N59/36 R-mAb project. (D) Agarose gel analysis of products of restriction enzyme digestion of N59/36 plasmid DNA with NotI and AscI. The plasmid backbone is seven kbp, and the intact insert comprising the VL and VH domains and the intervening joining fragment is 2.4 kbp. (E) Agarose gel analysis of PCR products of VL domain cDNA synthesized from RNA extracted from mouse splenocytes, the fusion partner Sp2/0-Ag14, and various hybridomas after digestion with the BciVI restriction enzyme to cleave the Sp2/0-Ag14-derived aberrant light chain product. The intact VL domains are » 360 bp, and the digested aberrant light chains » 180 bp. DOI: https://doi.org/10.7554/eLife.43322.004

    Journal: eLife

    Article Title: A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain

    doi: 10.7554/elife.43322

    Figure Lengend Snippet: Figure 2. Cloning of VL and VH domain sequences from hybridomas into the R-mAb expression plasmid. (A) Agarose gel analysis of VL and VH domain PCR products amplified from cDNA synthesized from RNA extracted from the N59/36 (anti-NR2B/GRIN2B) and K39/25 (anti-Kv2.1/KCNB1) hybridomas. The expected size of mouse IgG VL and VH domains is » 360 bp. (B) Agarose gel analysis of VH and digested VL fragments joined by fusion PCR (F-PCR) to the P1316-derived joining fragment to create a dual IgG chain cassette. (C) Agarose gel analysis of colony PCR samples of transformants from the N59/36 R-mAb project. (D) Agarose gel analysis of products of restriction enzyme digestion of N59/36 plasmid DNA with NotI and AscI. The plasmid backbone is seven kbp, and the intact insert comprising the VL and VH domains and the intervening joining fragment is 2.4 kbp. (E) Agarose gel analysis of PCR products of VL domain cDNA synthesized from RNA extracted from mouse splenocytes, the fusion partner Sp2/0-Ag14, and various hybridomas after digestion with the BciVI restriction enzyme to cleave the Sp2/0-Ag14-derived aberrant light chain product. The intact VL domains are » 360 bp, and the digested aberrant light chains » 180 bp. DOI: https://doi.org/10.7554/eLife.43322.004

    Article Snippet: Amplification of the Joining Fragment (Crosnier et al., 2010) Primer 21: 5’- GGGCTGATGCTGCACCAACTGTA-3’ Primer 26: 5’-ACTGCTTGAGGCTGGACTCGTGAACAATAGCAGC-3’ Colony PCR: UpNotI: 5’-TTTCAGACCCAGGTACTCAT-3’ DownAscI: 5’-GGGCAGCAGATCCAGGGGCC-3’ (reverse primer for IgG1 vector) Rev IgG2a: 5’- ACCCTTGACCAGGCATCCTAGAGT- 3’ (reverse primer for IgG2a vector) Mouse g2a CH domain amplification (restriction sites are underlined): IgG2a-F-AscI: 5’-ATATCACGGCGCGCCCAACAGCCCCATCGGTCTATCCA-3’ IgG2a-R-XbaI: 5’GACTGATCTAGATCATTTACCCGGAGTCCGGGAGAA-3’ R-mAb Sequencing: Forward strand of VL region: UpNotI = 5’-TTTCAGACCCAGGTACTCAT-3’ Reverse strand of VL region (IgG2a plasmids): Seq_VL_Rev_IgG2a = 5’ - CCAACTGTTCAG- GACGCCATT 3’ Forward strand of VH region: VH_seq_Forward = 5’- TCCCAGGCCACCATGAA 3’ Reverse strand of VH region (IgG1 plasmids): DownAscI = 5’-GGGCAGCAGATCCAGGGGCC-3’ Reverse strand of VH region (IgG2a plasmids): Rev IgG2a = 5’- ACCCTTGACCAGGCATCC TAGAGT- 3’ RNA preparation from cryopreserved hybridomas and RT-PCR The Ambion RNAqueous 96 Extraction kit (Thermo Fisher Cat# AM1920) was used for high throughput RNA extraction.

    Techniques: Cloning, Expressing, Plasmid Preparation, Agarose Gel Electrophoresis, Amplification, Synthesized, Derivative Assay

    Figure 3. Validation of subclass-switched anti-PSD-95 K28/43R R-mAb. (A) Validation of the K28/43R R-mAb in heterologous cells. COS-1 cells transiently transfected to express human PSD-95 in a subset of cells were immunolabeled with K28/43 mAb (IgG2a) alone (top row), K28/43R R-mAb (IgG1) alone (middle row), or K28/43 mAb plus K28/43R R-mAb (bottom row). Immunolabeling in all samples was detected with a cocktail of anti-mouse IgG2a (red, for the K28/43 mAb) and anti-mouse IgG1 (green, for the K28/43R R-mAb) subclass-specific Alexa Fluor conjugated secondary antibodies. Labeling in blue is for the DNA-specific dye Hoechst 33258 and shows nuclei of both transfected and untransfected cells. Scale bar in the lower right merged panel = 30 mm and holds for all panels in A. (B) Validation of the K28/43R R-mAb in brain sections. A brain section from an adult rat was immunolabeled with K28/43 mAb plus K28/43R R-mAb and immunolabeling detected with a cocktail of anti-mouse IgG2a (red, for K28/43 mAb) and anti-mouse IgG1 (green, for K28/43R R-mAb) subclass-specific Alexa Fluor conjugated secondary antibodies. Cell nuclei are labeled with the DNA- specific dye Hoechst 33258 (blue). The region of interest shown is from cerebellar cortex. Scale bar in the left panel = 100 mm, and in the right merged panel = 30 mm. (C) Immunoblots against brain membranes and COS cell lysates over-expressing various members of the MAGUK superfamily of scaffolding proteins. To confirm expression of MAGUK proteins, immunoblots were probed with rabbit anti-PSD-95 (red). K28/86 is an anti-MAGUK mAb. Primary antibodies were detected with the appropriate combinations of fluorescently labeled species-specific anti-rabbit and subclass-specific anti-mouse IgG secondary Abs as indicated. Control indicates COS cells transfected with an empty vector. DOI: https://doi.org/10.7554/eLife.43322.006

    Journal: eLife

    Article Title: A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain

    doi: 10.7554/elife.43322

    Figure Lengend Snippet: Figure 3. Validation of subclass-switched anti-PSD-95 K28/43R R-mAb. (A) Validation of the K28/43R R-mAb in heterologous cells. COS-1 cells transiently transfected to express human PSD-95 in a subset of cells were immunolabeled with K28/43 mAb (IgG2a) alone (top row), K28/43R R-mAb (IgG1) alone (middle row), or K28/43 mAb plus K28/43R R-mAb (bottom row). Immunolabeling in all samples was detected with a cocktail of anti-mouse IgG2a (red, for the K28/43 mAb) and anti-mouse IgG1 (green, for the K28/43R R-mAb) subclass-specific Alexa Fluor conjugated secondary antibodies. Labeling in blue is for the DNA-specific dye Hoechst 33258 and shows nuclei of both transfected and untransfected cells. Scale bar in the lower right merged panel = 30 mm and holds for all panels in A. (B) Validation of the K28/43R R-mAb in brain sections. A brain section from an adult rat was immunolabeled with K28/43 mAb plus K28/43R R-mAb and immunolabeling detected with a cocktail of anti-mouse IgG2a (red, for K28/43 mAb) and anti-mouse IgG1 (green, for K28/43R R-mAb) subclass-specific Alexa Fluor conjugated secondary antibodies. Cell nuclei are labeled with the DNA- specific dye Hoechst 33258 (blue). The region of interest shown is from cerebellar cortex. Scale bar in the left panel = 100 mm, and in the right merged panel = 30 mm. (C) Immunoblots against brain membranes and COS cell lysates over-expressing various members of the MAGUK superfamily of scaffolding proteins. To confirm expression of MAGUK proteins, immunoblots were probed with rabbit anti-PSD-95 (red). K28/86 is an anti-MAGUK mAb. Primary antibodies were detected with the appropriate combinations of fluorescently labeled species-specific anti-rabbit and subclass-specific anti-mouse IgG secondary Abs as indicated. Control indicates COS cells transfected with an empty vector. DOI: https://doi.org/10.7554/eLife.43322.006

    Article Snippet: Amplification of the Joining Fragment (Crosnier et al., 2010) Primer 21: 5’- GGGCTGATGCTGCACCAACTGTA-3’ Primer 26: 5’-ACTGCTTGAGGCTGGACTCGTGAACAATAGCAGC-3’ Colony PCR: UpNotI: 5’-TTTCAGACCCAGGTACTCAT-3’ DownAscI: 5’-GGGCAGCAGATCCAGGGGCC-3’ (reverse primer for IgG1 vector) Rev IgG2a: 5’- ACCCTTGACCAGGCATCCTAGAGT- 3’ (reverse primer for IgG2a vector) Mouse g2a CH domain amplification (restriction sites are underlined): IgG2a-F-AscI: 5’-ATATCACGGCGCGCCCAACAGCCCCATCGGTCTATCCA-3’ IgG2a-R-XbaI: 5’GACTGATCTAGATCATTTACCCGGAGTCCGGGAGAA-3’ R-mAb Sequencing: Forward strand of VL region: UpNotI = 5’-TTTCAGACCCAGGTACTCAT-3’ Reverse strand of VL region (IgG2a plasmids): Seq_VL_Rev_IgG2a = 5’ - CCAACTGTTCAG- GACGCCATT 3’ Forward strand of VH region: VH_seq_Forward = 5’- TCCCAGGCCACCATGAA 3’ Reverse strand of VH region (IgG1 plasmids): DownAscI = 5’-GGGCAGCAGATCCAGGGGCC-3’ Reverse strand of VH region (IgG2a plasmids): Rev IgG2a = 5’- ACCCTTGACCAGGCATCC TAGAGT- 3’ RNA preparation from cryopreserved hybridomas and RT-PCR The Ambion RNAqueous 96 Extraction kit (Thermo Fisher Cat# AM1920) was used for high throughput RNA extraction.

    Techniques: Biomarker Discovery, Transfection, Immunolabeling, Labeling, Western Blot, Expressing, Scaffolding, Control, Plasmid Preparation

    Figure 4. Multiplex immunolabeling with subclass-switched recombinant antibodies in adult rat brain. (A) A section from neocortex labeled with anti- pan-Nav R-mAb K58/35R (IgG2a, red) at nodes of Ranvier and AIS (arrows), anti-CASPR mAb K65/35 (IgG1, green) at paranodes, and anti-Kv2.1 rabbit polyclonal (KC) antibody (blue) on somata and proximal dendrites. Scale bar = 150 mm. Insets (dashed box) show details of labeling for pan-Nav (red) and CASPR (green) at the node and paranodes (arrows), respectively, at a single node of Ranvier as indicated by box in main panel. (B) A section Figure 4 continued on next page

    Journal: eLife

    Article Title: A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain

    doi: 10.7554/elife.43322

    Figure Lengend Snippet: Figure 4. Multiplex immunolabeling with subclass-switched recombinant antibodies in adult rat brain. (A) A section from neocortex labeled with anti- pan-Nav R-mAb K58/35R (IgG2a, red) at nodes of Ranvier and AIS (arrows), anti-CASPR mAb K65/35 (IgG1, green) at paranodes, and anti-Kv2.1 rabbit polyclonal (KC) antibody (blue) on somata and proximal dendrites. Scale bar = 150 mm. Insets (dashed box) show details of labeling for pan-Nav (red) and CASPR (green) at the node and paranodes (arrows), respectively, at a single node of Ranvier as indicated by box in main panel. (B) A section Figure 4 continued on next page

    Article Snippet: Amplification of the Joining Fragment (Crosnier et al., 2010) Primer 21: 5’- GGGCTGATGCTGCACCAACTGTA-3’ Primer 26: 5’-ACTGCTTGAGGCTGGACTCGTGAACAATAGCAGC-3’ Colony PCR: UpNotI: 5’-TTTCAGACCCAGGTACTCAT-3’ DownAscI: 5’-GGGCAGCAGATCCAGGGGCC-3’ (reverse primer for IgG1 vector) Rev IgG2a: 5’- ACCCTTGACCAGGCATCCTAGAGT- 3’ (reverse primer for IgG2a vector) Mouse g2a CH domain amplification (restriction sites are underlined): IgG2a-F-AscI: 5’-ATATCACGGCGCGCCCAACAGCCCCATCGGTCTATCCA-3’ IgG2a-R-XbaI: 5’GACTGATCTAGATCATTTACCCGGAGTCCGGGAGAA-3’ R-mAb Sequencing: Forward strand of VL region: UpNotI = 5’-TTTCAGACCCAGGTACTCAT-3’ Reverse strand of VL region (IgG2a plasmids): Seq_VL_Rev_IgG2a = 5’ - CCAACTGTTCAG- GACGCCATT 3’ Forward strand of VH region: VH_seq_Forward = 5’- TCCCAGGCCACCATGAA 3’ Reverse strand of VH region (IgG1 plasmids): DownAscI = 5’-GGGCAGCAGATCCAGGGGCC-3’ Reverse strand of VH region (IgG2a plasmids): Rev IgG2a = 5’- ACCCTTGACCAGGCATCC TAGAGT- 3’ RNA preparation from cryopreserved hybridomas and RT-PCR The Ambion RNAqueous 96 Extraction kit (Thermo Fisher Cat# AM1920) was used for high throughput RNA extraction.

    Techniques: Multiplex Assay, Immunolabeling, Recombinant, Labeling

    Figure 5. Cloning of anti-Kv2.1 D3/71 VL and VH domain cDNAs from a nonviable hybridoma. (A) Agarose gel analysis of PCR amplified VL and VH domains from cDNA synthesized from RNA extracted from the non-viable D3/71 hybridoma. The panel to the right shows the VL after digestion with the BciVI restriction enzyme to cleave the Sp2/0-Ag14-derived aberrant light chain product. The expected size of mouse IgG VL and VH domains is » 360 bp, and of the cleaved aberrant VL domain is » 180 bp. (B) Agarose gel analysis of D3/71 VH and digested VL fragments joined by fusion PCR (F- PCR) to the P1316 joining fragment to create a dual IgG chain cassette. (C) Agarose gel analysis of colony PCR samples of transformants from the of D3/71 R-mAb project. (D) Agarose gel analysis of products of restriction enzyme digestion of D3/71 plasmid DNA with NotI and AscI. The plasmid backbone is seven kbp, and the intact insert comprising the VL and VH domains and the intervening joining fragment is 2.4 kbp. DOI: https://doi.org/10.7554/eLife.43322.008

    Journal: eLife

    Article Title: A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain

    doi: 10.7554/elife.43322

    Figure Lengend Snippet: Figure 5. Cloning of anti-Kv2.1 D3/71 VL and VH domain cDNAs from a nonviable hybridoma. (A) Agarose gel analysis of PCR amplified VL and VH domains from cDNA synthesized from RNA extracted from the non-viable D3/71 hybridoma. The panel to the right shows the VL after digestion with the BciVI restriction enzyme to cleave the Sp2/0-Ag14-derived aberrant light chain product. The expected size of mouse IgG VL and VH domains is » 360 bp, and of the cleaved aberrant VL domain is » 180 bp. (B) Agarose gel analysis of D3/71 VH and digested VL fragments joined by fusion PCR (F- PCR) to the P1316 joining fragment to create a dual IgG chain cassette. (C) Agarose gel analysis of colony PCR samples of transformants from the of D3/71 R-mAb project. (D) Agarose gel analysis of products of restriction enzyme digestion of D3/71 plasmid DNA with NotI and AscI. The plasmid backbone is seven kbp, and the intact insert comprising the VL and VH domains and the intervening joining fragment is 2.4 kbp. DOI: https://doi.org/10.7554/eLife.43322.008

    Article Snippet: Amplification of the Joining Fragment (Crosnier et al., 2010) Primer 21: 5’- GGGCTGATGCTGCACCAACTGTA-3’ Primer 26: 5’-ACTGCTTGAGGCTGGACTCGTGAACAATAGCAGC-3’ Colony PCR: UpNotI: 5’-TTTCAGACCCAGGTACTCAT-3’ DownAscI: 5’-GGGCAGCAGATCCAGGGGCC-3’ (reverse primer for IgG1 vector) Rev IgG2a: 5’- ACCCTTGACCAGGCATCCTAGAGT- 3’ (reverse primer for IgG2a vector) Mouse g2a CH domain amplification (restriction sites are underlined): IgG2a-F-AscI: 5’-ATATCACGGCGCGCCCAACAGCCCCATCGGTCTATCCA-3’ IgG2a-R-XbaI: 5’GACTGATCTAGATCATTTACCCGGAGTCCGGGAGAA-3’ R-mAb Sequencing: Forward strand of VL region: UpNotI = 5’-TTTCAGACCCAGGTACTCAT-3’ Reverse strand of VL region (IgG2a plasmids): Seq_VL_Rev_IgG2a = 5’ - CCAACTGTTCAG- GACGCCATT 3’ Forward strand of VH region: VH_seq_Forward = 5’- TCCCAGGCCACCATGAA 3’ Reverse strand of VH region (IgG1 plasmids): DownAscI = 5’-GGGCAGCAGATCCAGGGGCC-3’ Reverse strand of VH region (IgG2a plasmids): Rev IgG2a = 5’- ACCCTTGACCAGGCATCC TAGAGT- 3’ RNA preparation from cryopreserved hybridomas and RT-PCR The Ambion RNAqueous 96 Extraction kit (Thermo Fisher Cat# AM1920) was used for high throughput RNA extraction.

    Techniques: Cloning, Agarose Gel Electrophoresis, Amplification, Synthesized, Derivative Assay, Plasmid Preparation

    Figure 6. Recovery of a functional anti-Kv2.1 D3/71R R-mAb from nonviable hybridomas. (A) Validation of the D3/71R R-mAb in heterologous cells. COS-1 cells transiently transfected to express rat Kv2.1 in a subset of cells were immunolabeled with K89/34 mAb (IgG1) alone (top row), D3/71R R-mAb (IgG2a) alone (middle row), or K89/34 mAb plus D3/71R R-mAb (bottom row). Immunolabeling in all samples was detected with a cocktail of anti-mouse IgG1 (green, for the K89/34 mAb) and anti-mouse IgG2a (red, for the D3/71R R-mAb) subclass-specific Alexa Fluor conjugated secondary antibodies. (B) Validation of the subclass-switched K89/34R R-mAb in heterologous cells. COS-1 cells transiently transfected to express rat Kv2.1 in a subset of cells were immunolabeled with K89/34 mAb (IgG1) alone (top row), K89/34R R-mAb (IgG2a) alone (middle row), or K89/34 mAb plus K89/34R R-mAb (bottom row). Immunolabeling in all samples was detected with a cocktail of anti-mouse IgG1 (green, for the K89/34 mAb) and anti-mouse IgG2a (red, for the K89/34R R-mAb) subclass-specific Alexa Fluor conjugated secondary antibodies. Labeling in blue in panels A and B is for the DNA-specific dye Hoechst 33258 and shows nuclei of both transfected and untransfected cells. Scale bar in the lower right merged panel = 30 mm and holds for all panels in A and B. (C) Validation of the D3/71R R-mAb in brain sections. A brain section from an adult rat was immunolabeled with K89/34 mAb plus D3/71 R-mAb and the immunolabeling detected with a cocktail of anti-mouse IgG1 (green, for the K89/34 mAb) and anti-mouse IgG2a (red, for the D3/71R R-mAb) subclass-specific Alexa Fluor conjugated secondary antibodies. Cell nuclei are labeled with the DNA-specific dye Hoechst 33258 (blue). Region of interest shown is from neocortex. Scale bar = 30 mm. (D) Strip immunoblots on a crude rat brain membrane fraction immunolabeled with the K89/34 mAb, the K89/34R R-mAb, and the D3/71 R-mAb as indicated. Immunolabeling was detected on autoradiography film after treatment of strip immunoblots with HRP-conjugated anti-mouse IgG-specific secondary antibody and ECL. DOI: https://doi.org/10.7554/eLife.43322.009

    Journal: eLife

    Article Title: A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain

    doi: 10.7554/elife.43322

    Figure Lengend Snippet: Figure 6. Recovery of a functional anti-Kv2.1 D3/71R R-mAb from nonviable hybridomas. (A) Validation of the D3/71R R-mAb in heterologous cells. COS-1 cells transiently transfected to express rat Kv2.1 in a subset of cells were immunolabeled with K89/34 mAb (IgG1) alone (top row), D3/71R R-mAb (IgG2a) alone (middle row), or K89/34 mAb plus D3/71R R-mAb (bottom row). Immunolabeling in all samples was detected with a cocktail of anti-mouse IgG1 (green, for the K89/34 mAb) and anti-mouse IgG2a (red, for the D3/71R R-mAb) subclass-specific Alexa Fluor conjugated secondary antibodies. (B) Validation of the subclass-switched K89/34R R-mAb in heterologous cells. COS-1 cells transiently transfected to express rat Kv2.1 in a subset of cells were immunolabeled with K89/34 mAb (IgG1) alone (top row), K89/34R R-mAb (IgG2a) alone (middle row), or K89/34 mAb plus K89/34R R-mAb (bottom row). Immunolabeling in all samples was detected with a cocktail of anti-mouse IgG1 (green, for the K89/34 mAb) and anti-mouse IgG2a (red, for the K89/34R R-mAb) subclass-specific Alexa Fluor conjugated secondary antibodies. Labeling in blue in panels A and B is for the DNA-specific dye Hoechst 33258 and shows nuclei of both transfected and untransfected cells. Scale bar in the lower right merged panel = 30 mm and holds for all panels in A and B. (C) Validation of the D3/71R R-mAb in brain sections. A brain section from an adult rat was immunolabeled with K89/34 mAb plus D3/71 R-mAb and the immunolabeling detected with a cocktail of anti-mouse IgG1 (green, for the K89/34 mAb) and anti-mouse IgG2a (red, for the D3/71R R-mAb) subclass-specific Alexa Fluor conjugated secondary antibodies. Cell nuclei are labeled with the DNA-specific dye Hoechst 33258 (blue). Region of interest shown is from neocortex. Scale bar = 30 mm. (D) Strip immunoblots on a crude rat brain membrane fraction immunolabeled with the K89/34 mAb, the K89/34R R-mAb, and the D3/71 R-mAb as indicated. Immunolabeling was detected on autoradiography film after treatment of strip immunoblots with HRP-conjugated anti-mouse IgG-specific secondary antibody and ECL. DOI: https://doi.org/10.7554/eLife.43322.009

    Article Snippet: Amplification of the Joining Fragment (Crosnier et al., 2010) Primer 21: 5’- GGGCTGATGCTGCACCAACTGTA-3’ Primer 26: 5’-ACTGCTTGAGGCTGGACTCGTGAACAATAGCAGC-3’ Colony PCR: UpNotI: 5’-TTTCAGACCCAGGTACTCAT-3’ DownAscI: 5’-GGGCAGCAGATCCAGGGGCC-3’ (reverse primer for IgG1 vector) Rev IgG2a: 5’- ACCCTTGACCAGGCATCCTAGAGT- 3’ (reverse primer for IgG2a vector) Mouse g2a CH domain amplification (restriction sites are underlined): IgG2a-F-AscI: 5’-ATATCACGGCGCGCCCAACAGCCCCATCGGTCTATCCA-3’ IgG2a-R-XbaI: 5’GACTGATCTAGATCATTTACCCGGAGTCCGGGAGAA-3’ R-mAb Sequencing: Forward strand of VL region: UpNotI = 5’-TTTCAGACCCAGGTACTCAT-3’ Reverse strand of VL region (IgG2a plasmids): Seq_VL_Rev_IgG2a = 5’ - CCAACTGTTCAG- GACGCCATT 3’ Forward strand of VH region: VH_seq_Forward = 5’- TCCCAGGCCACCATGAA 3’ Reverse strand of VH region (IgG1 plasmids): DownAscI = 5’-GGGCAGCAGATCCAGGGGCC-3’ Reverse strand of VH region (IgG2a plasmids): Rev IgG2a = 5’- ACCCTTGACCAGGCATCC TAGAGT- 3’ RNA preparation from cryopreserved hybridomas and RT-PCR The Ambion RNAqueous 96 Extraction kit (Thermo Fisher Cat# AM1920) was used for high throughput RNA extraction.

    Techniques: Functional Assay, Biomarker Discovery, Transfection, Immunolabeling, Labeling, Stripping Membranes, Western Blot, Membrane, Autoradiography