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: ( A ) Agarose gel analysis of V L and V H 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 V L and V H domains is ≈360 bp. ( B ) Agarose gel analysis of V H and digested V L 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 V L and V H domains and the intervening joining fragment is 2.4 kbp. ( E ) Agarose gel analysis of PCR products of V L 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 V L domains are ≈360 bp, and the digested aberrant light chains ≈180 bp.

Article Snippet: The IgG2a C H PCR product and the K28/43 IgG1 recombinant plasmid were both digested with AscI and XbaI restriction enzymes (New England BioLabs Cat# R0558 and R0145, respectively) and column purified (Qiagen/QiaQuick PCR Purification Cat# 28106) or agarose gel purified, respectively (Qiagen/QiaQuick Gel Extraction Cat# 28706).

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

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: ( 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 µm 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 µm, and in the right merged panel = 30 µm. ( 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.

Article Snippet: The IgG2a C H PCR product and the K28/43 IgG1 recombinant plasmid were both digested with AscI and XbaI restriction enzymes (New England BioLabs Cat# R0558 and R0145, respectively) and column purified (Qiagen/QiaQuick PCR Purification Cat# 28106) or agarose gel purified, respectively (Qiagen/QiaQuick Gel Extraction Cat# 28706).

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

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: ( 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 μm. 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 through cerebellum showing labeling with anti-GABA-AR β1 R-mAb N96/55R (IgG2a, magenta) in the molecular layer (ML), and anti-GABA-AR β3 mAb N87/25 (IgG1, green) in the granule cell layer (GL). PCL = Purkinje cell layer. Scale bar = 150 μm. ( C ) A section through hippocampus labeled with anti-Kv2.1 R-mAb 89/34R (IgG2a, red) on somata and proximal dendrites, anti-AnkyrinG mAb N106/65 (IgG2b, green) on AIS, and nuclear stain Hoechst 33258 (blue). Scale bar = 150 μm. Panels C1-C3 show magnified details of labeling for pan-Kv2.1 (red) on somata and proximal dendrites (arrows in C1), and anti-AnkyrinG (green) on AIS (arrows in C2). Scale bar = 50 μm ( C1–C3 ). ( D ) A section through cerebellum labelled with anti-GABA-AR α6 R-mAb K229A/32R (IgG2a, red) in the granule cell layer (GL), anti-pan-QKI mAb N147/6 (IgG2b, green) labeling glial cells in/near the Purkinje cell layer (PCL), and anti-Kv4.2 mAb K57/1 (IgG1, blue) labeling the granule cell layer (GL). Scale bar = 30 μm. ( E ) A section from neocortex labelled with anti-Kv2.1 R-mAb 89/34R (IgG2a, red) on somata and proximal dendrites of neurons, and anti-GFAP mAb N206A/8 (IgG1, green) and anti-pan-QKI mAb N147/6 (IgG2b, blue) labeling glial cell processes and cell bodies respectively. Scale bar = 15 μm. ( F ) A section through cerebellum showing labeling with anti-PSD-93 R-mAb N18/30R (IgG2a, red) in the cell bodies and dendrites of Purkinje cells, the nuclear stain Hoechst 33258 (green) and anti-VGluT1 mAb N28/9 (IgG1, blue) in the molecular layer (ML). PCL = Purkinje cell layer. Scale bar = 10 μm.

Article Snippet: The IgG2a C H PCR product and the K28/43 IgG1 recombinant plasmid were both digested with AscI and XbaI restriction enzymes (New England BioLabs Cat# R0558 and R0145, respectively) and column purified (Qiagen/QiaQuick PCR Purification Cat# 28106) or agarose gel purified, respectively (Qiagen/QiaQuick Gel Extraction Cat# 28706).

Techniques: Labeling, Staining

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: ( A ) Agarose gel analysis of PCR amplified V L and V H domains from cDNA synthesized from RNA extracted from the non-viable D3/71 hybridoma. The panel to the right shows the V L after digestion with the BciVI restriction enzyme to cleave the Sp2/0-Ag14-derived aberrant light chain product. The expected size of mouse IgG V L and V H domains is ≈360 bp, and of the cleaved aberrant V L domain is ≈180 bp. ( B ) Agarose gel analysis of D3/71 V H and digested V L 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 V L and V H domains and the intervening joining fragment is 2.4 kbp.

Article Snippet: The IgG2a C H PCR product and the K28/43 IgG1 recombinant plasmid were both digested with AscI and XbaI restriction enzymes (New England BioLabs Cat# R0558 and R0145, respectively) and column purified (Qiagen/QiaQuick PCR Purification Cat# 28106) or agarose gel purified, respectively (Qiagen/QiaQuick Gel Extraction Cat# 28706).

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

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: ( 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 µm 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 µm. ( 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.

Article Snippet: The IgG2a C H PCR product and the K28/43 IgG1 recombinant plasmid were both digested with AscI and XbaI restriction enzymes (New England BioLabs Cat# R0558 and R0145, respectively) and column purified (Qiagen/QiaQuick PCR Purification Cat# 28106) or agarose gel purified, respectively (Qiagen/QiaQuick Gel Extraction Cat# 28706).

Techniques: Transfection, Immunolabeling, Labeling, Stripping Membranes, Western Blot, Autoradiography

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: Non-R-mAb antibodies used in this study. Table lists Abs used in this study outside of the R-mAbs whose generation is described here. For each Ab the name, immunogen used in Ab generation, source and RRID number in the Antibody Registry, form and concentration/dilution used, and specific use in this paper is detailed.

Article Snippet: The IgG2a C H PCR product and the K28/43 IgG1 recombinant plasmid were both digested with AscI and XbaI restriction enzymes (New England BioLabs Cat# R0558 and R0145, respectively) and column purified (Qiagen/QiaQuick PCR Purification Cat# 28106) or agarose gel purified, respectively (Qiagen/QiaQuick Gel Extraction Cat# 28706).

Techniques: Concentration Assay, Affinity Purification, Recombinant

Journal: The Journal of Neuroscience

Article Title: Spinal Wnt5a Plays a Key Role in Spinal Dendritic Spine Remodeling in Neuropathic and Inflammatory Pain Models and in the Proalgesic Effects of Peripheral Wnt3a

doi: 10.1523/JNEUROSCI.2942-19.2020

Figure Lengend Snippet: Noncanonical Ror2 and Ryk receptors mediate spinal Wnt5a-induced nociceptive hypersensitivity. A, B, Typical example of coimmunoreactivity for Ror2 (A) or Ryk (B; both in red) in the spinal dorsal horn, together with the markers for excitatory synapses (PSD-95, left) or inhibitory synapses (Gephyrin, right; both in green). Bottom panels show negative controls. Scale bar, 10 μm. C, D, Evidence for marked RNA interference-mediated knockdown of Ror2 (C) or Ryk (D) mRNA in the spinal dorsal horn on intrathecal injection of siRNA against Ror2 (si-Ror2) or Ryk (si-Ryk), respectively, in relation to intrathecal injection of the respective scrambled siRNAs as controls (si-Scramble). N= 4 mice/condition. Student's t test was performed. *p < 0.05 compared with the corresponding control group. E, H, Complete blockade of Wnt5a-induced mechanical hypersensitivity in mice intrathecally injected with siRNA against Ror2 (E, F) or siRNA against RyK (G, H) compared with mice injected with scrambled siRNA (n = 6–7 mice/group). Stimulus–response curves of the one paw are shown in E and G, whereas their integral over time (AUC) are shown in F and H. Two-way ANOVA followed by Tukey's test was performed. *p < 0.05 compared with the corresponding control group; †p < 0.05 compared with si-Scramble + Wnt5a-treated animals. In all panels, data are represented as the mean ± SEM, and bar graphs are also shown as scatter plots.

Article Snippet: Following Na-Citrate antigen retrieval treatment, the sections were stained with the following antibodies: sheep anti-Ryk (1:25; catalog #AF4649, R&D Systems), rabbit anti-Ror2 (1:100; catalog #PA5-77 028, Thermo Fisher Scientific), rabbit anti-PSD-95 (1:100; catalog #51–6900, Thermo Fisher Scientific), mouse anti-PSD-95 (1:100; anti-PSD-95 MAGUK scaffold protein, NeuroMab clone K28/74, catalog #75–348, UC Davis/NIH NeuroMab Facility; RRID: AB_2315909 ), mouse anti-Gephyrin (1:500; catalog #147021, SySy), and rabbit anti-Gephyrin (1:1000; catalog #147018, SySy).

Techniques: Injection