Journal: Nature Communications

Article Title: Proteomic and functional analyses of the periodic membrane skeleton in neurons

doi: 10.1038/s41467-022-30720-x

Figure Lengend Snippet: a Left: 3D STORM images of two potassium channel subunits, K v 1.2 and K v 1.3, in axons. Middle: 1D autocorrelation of the imaged proteins for the axon region indicated by the dashed line in the left panels. Right: Average 1D autocorrelation of the imaged proteins over 20–90 randomly chosen axon regions. b Same as ( a ) but for five cell adhesion molecules, including neurofascin, NrCAM, L1CAM, NCAM1 and CHL1. c Same as ( a ) but for three membrane-associated (non-transmembrane) signaling molecules, including calcium/calmodulin-dependent protein kinase type IIβ (CAMK IIβ), heterotrimeric G protein β-subunit 1, and brain acid soluble protein 1, as well as a transmembrane signaling molecule, glycoprotein M6A. STORM images in a – c are representative examples from three independent experiments with similar results. Scale bars: 1 μm. Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used in this study: guinea pig anti-MAP2 antibody 1:500 dilution for immunofluorescence (IF) (Synaptic Systems, 188004), rabbit anti-MAP2 antibody 1:500 for IF (Synaptic Systems, 188002), mouse anti-αII spectrin antibody 1:400 for IF (Biolegend, 803201, Clone D8B7), mouse anti-αII spectrin antibody 1:200 for IF (Encor Biotechnology, MCA-3D7, Clone 3D7), rabbit anti-αII spectrin antibody 1:200 for IF (Encor Biotechnology, RPCA-aII-Spec), mouse anti-αII spectrin antibody 1:200 for IF (EMD Millipore, MAB1622, Clone AA6), mouse anti-βII spectrin antibody 1:200 for IF (BD Biosciences, 612563, Clone 42), mouse anti-dematin antibody 1:50 for IF (Santa Cruz Biotechnology, sc-135881, Clone 18), rabbit anti-coronin 2B antibody 1:200 for IF (Novus Biologicals, NBP 1-85567), mouse anti-tubulin antibody 1:100 for IF (Santa Cruz Biotechnology, sc-5286, Clone B7), rabbit anti-Tau antibody 1:500 for IF (Synaptic Systems, 314002), mouse anti-K v 1.2 channel antibody 1:200 for IF (Neuromab, 75-008, Clone K14/16), rabbit anti-neurofascin antibody 1:200 for IF (Neuromab, 75–172, Clone A12/18), rabbit anti-NrCAM 1:200 for IF (Abcam, ab24344), goat anti-CHL1 antibody 1:200 for IF (R&D systems, AF2147), rabbit anti-NCAM1 antibody 1:200 for IF (EMD Millipore, AB5032), mouse anti-ankyrin G antibody 1:100 for IF (Santa Cruz Biotechnology, sc-12719, Clone 463), mouse anti-bassoon antibody 1:400 for IF (Enzo, ADI-VAM-PS003-F, Clone SAP7F407), rabbit anti-homer antibody 1:500 for IF (Synaptic Systems, 160003), rabbit anti-L1CAM antibody 1:500 for Western blot (WB) (ABclonal, A8555), rat anti-L1CAM antibody 1:200 for IF (R&D Systems, MAB5674, Clone 555), rabbit anti-NMIIB (Myh10) (N-terminus) antibody 1:200 for IF (GeneTex, GTX133378), rabbit anti-NMIIA (Myh9) (N-terminus) antibody 1:200 for IF (GeneTex, GTX101751), rabbit anti-NMIIB (Myh10) (C-terminus) antibody 1:200 for IF (Biolegend, 909901), rabbit anti-Glutamate Receptor 2 & 3 antibody 1:200 for IF (EMD Millipore, AB1506), rabbit anti-GFP antibody 1:400 for IF (Thermo Fisher Scientific, A11122). rabbit anti-β-actin antibody 1:1000 for WB (Proteintech, 20536-1-AP).

Techniques: Membrane

Journal: Nature Communications

Article Title: Proteomic and functional analyses of the periodic membrane skeleton in neurons

doi: 10.1038/s41467-022-30720-x

Figure Lengend Snippet: a Top panels: Conventional fluorescence images of tubulin (green) and dendrite marker MAP2 (magenta) for neurons transfected with adenoviruses expressing scrambled (control) shRNA, βII-spectrin shRNA, ankyrin B shRNA, L1CAM shRNA, NCAM1 shRNA, CHL1 shRNA, or shRNAs against all three adhesion molecules (L1CAM, NCAM1 and CHL1). The tubulin-positive neurites lacking MAP2 signal are axons. Bottom panel: Average diameter of axon bundles (or single axons) quantified for the conditions described in the top panels. The last bar and dashed line represent the average single-axon diameter obtained from images of sparsely cultured neurons. * indicates p < 0.05 (two-sided unpaired student’s t-test); p-value s (from left to right): 3.9 × 10 −2 , 1.3 × 10 −2 , 1.7 × 10 −2 , 3.7 × 10 −2 , 1.8 × 10 −2 and 9.3 × 10 −3 . b Top: Conventional fluorescence images of cultured neurons immunostained for axon marker Tau (green) and dendrite marker MAP2 (magenta) under the conditions described in ( a ). Bottom: Average fraction of the total length of dendrites that are bundled with axons, quantified for the conditions indicated in the top panel. * p < 0.05 (two-sided unpaired student’s t-test); p-value s (from left to right): 1.7 × 10 −2 , 1.8 × 10 −2 , 4.9 × 10 −2 , 2.4 × 10 −2 and 1.5 × 10 −2 . c Top: Conventional fluorescence images of cultured neurons immunostained for the presynaptic marker Bassoon (green) and postsynaptic marker Homer1 (magenta) under the conditions described in ( a ). Bottom: Average synapse density per unit area on dendrites, quantified for the conditions as described in the top panel. Only puncta showing both presynaptic and postsynaptic marker signals were counted as synapses. * p < 0.05 and ** p < 0.005 (two-sided unpaired student’s t-test); p-value s (from left to right): 3.8 × 10 −2 , 6.4 × 10 −3 , 9.6 × 10 −3 and 3.3 × 10 −3 . Scale bars: 5 μm. Data are mean ± s.e.m ( n = 3 biological replicates; 15–25 imaged regions per condition). Images in a – c are representative examples from three independent experiments with similar results. Source data are provided in the Source Data file.

Article Snippet: The following primary antibodies were used in this study: guinea pig anti-MAP2 antibody 1:500 dilution for immunofluorescence (IF) (Synaptic Systems, 188004), rabbit anti-MAP2 antibody 1:500 for IF (Synaptic Systems, 188002), mouse anti-αII spectrin antibody 1:400 for IF (Biolegend, 803201, Clone D8B7), mouse anti-αII spectrin antibody 1:200 for IF (Encor Biotechnology, MCA-3D7, Clone 3D7), rabbit anti-αII spectrin antibody 1:200 for IF (Encor Biotechnology, RPCA-aII-Spec), mouse anti-αII spectrin antibody 1:200 for IF (EMD Millipore, MAB1622, Clone AA6), mouse anti-βII spectrin antibody 1:200 for IF (BD Biosciences, 612563, Clone 42), mouse anti-dematin antibody 1:50 for IF (Santa Cruz Biotechnology, sc-135881, Clone 18), rabbit anti-coronin 2B antibody 1:200 for IF (Novus Biologicals, NBP 1-85567), mouse anti-tubulin antibody 1:100 for IF (Santa Cruz Biotechnology, sc-5286, Clone B7), rabbit anti-Tau antibody 1:500 for IF (Synaptic Systems, 314002), mouse anti-K v 1.2 channel antibody 1:200 for IF (Neuromab, 75-008, Clone K14/16), rabbit anti-neurofascin antibody 1:200 for IF (Neuromab, 75–172, Clone A12/18), rabbit anti-NrCAM 1:200 for IF (Abcam, ab24344), goat anti-CHL1 antibody 1:200 for IF (R&D systems, AF2147), rabbit anti-NCAM1 antibody 1:200 for IF (EMD Millipore, AB5032), mouse anti-ankyrin G antibody 1:100 for IF (Santa Cruz Biotechnology, sc-12719, Clone 463), mouse anti-bassoon antibody 1:400 for IF (Enzo, ADI-VAM-PS003-F, Clone SAP7F407), rabbit anti-homer antibody 1:500 for IF (Synaptic Systems, 160003), rabbit anti-L1CAM antibody 1:500 for Western blot (WB) (ABclonal, A8555), rat anti-L1CAM antibody 1:200 for IF (R&D Systems, MAB5674, Clone 555), rabbit anti-NMIIB (Myh10) (N-terminus) antibody 1:200 for IF (GeneTex, GTX133378), rabbit anti-NMIIA (Myh9) (N-terminus) antibody 1:200 for IF (GeneTex, GTX101751), rabbit anti-NMIIB (Myh10) (C-terminus) antibody 1:200 for IF (Biolegend, 909901), rabbit anti-Glutamate Receptor 2 & 3 antibody 1:200 for IF (EMD Millipore, AB1506), rabbit anti-GFP antibody 1:400 for IF (Thermo Fisher Scientific, A11122). rabbit anti-β-actin antibody 1:1000 for WB (Proteintech, 20536-1-AP).

Techniques: Fluorescence, Marker, Transfection, Expressing, Control, shRNA, Cell Culture

Journal: bioRxiv

Article Title: Alteration of nociceptive Schwann cells in a mouse model of peripheral neuropathy in prediabetic condition

doi: 10.1101/2024.03.12.584541

Figure Lengend Snippet: (A) PGP9.5 immunofluorescence with DAPI staining in foot skin section of control (n=5) and HFD-fed mice (n=5). (B) Quantitation of IENFD is presented as the number of fibers/mm of epidermis. (C) L1CAM immunofluorescence with DAPI staining in foot skin section of control (n=5) and HFD-fed mice (n=5). Arrowhead indicate nerve fibers in the epidermis of the foot skin. Arrows indicate nociceptive Schwann cells and their cellular extensions at the border of the epidermis and the dermis. (D) Mean fluorescence intensity quantification of L1CAM immunostaining at the localization of nociceptive Schwann cells (at the border of epidermis and dermis). (E) Quantification of L1CAM-positive cells and their cellular extensions presented in number/mm of epidermis. ** P < 0.01, *** P < 0.001: control diet vs. HFD. Data are presented as means ±SEM. Scale bar: 50 μm.

Article Snippet: The same protocol was applied for L1 cell adhesion molecule (L1CAM, 1:500, #20659-1-AP, ProteinTech) antibody but the sections were incubated only overnight at 4°C.

Techniques: Immunofluorescence, Staining, Control, Quantitation Assay, Fluorescence, Immunostaining

Journal: bioRxiv

Article Title: Diversification of multipotential postmitotic mouse retinal ganglion cell precursors into discrete types

doi: 10.1101/2021.10.21.465277

Figure Lengend Snippet: a. Retinal sections stained with L1CAM (red), which was used to enrich for RGCs at early stages, and the pan-RGC marker RBPMS , (green) at E13, E14, E16 and P0. Nuclei are counterstained by the Hoeschst dye (blue). b. Relative proportions (y-axis) of major cell classes shown in at each combination of age and enrichment method. Both anti-Thy1 and anti-L1cam were used to enrich RGCs at E13, E14, E16 and P0, but only anti-Thy1 was used at P5, because L1cam becomes localized to axons postnatally. AC, Amacrine Cells; RPC, retinal progenitor cells. c. Box and whisker plots show gene expression levels of key markers by RGCs as a function of age and enrichment method. Markers shown shown are two pan-RGC markers, Rbpms, Nefl , and the two cell-surface proteins used for enrichment, Thy1 and L1cam . Note that Thy1 expression is poor at E13, consistent with low RGC yield in anti-Thy1 enriched cells (panel B). Black horizontal line, median; bars, interquartile range; vertical lines, range; dots, outliers. d. Dotplot showing genes (columns) that are selectively expressed in RGCs and RPCs. The size of each circle is proportional to the percentage of cells expressing the gene, and the color depicts the average log-normalized expression. e. Co-embedding analysis of E14, E16 and P0 data collected in this study with whole retina single-cell transcriptomes in independent studies: E14, E16 and P0 data from 31 and E15.5 data from 43 . Cells (points) are visualized in UMAP and colored by study of origin. f. Same as e, with cells colored by the expression level of Nefl , an RGC marker. This shows the higher enrichment of RGCs in our study compared to 31 and 43 . g. Same as d, with cells colored by expression level of Fgf15 , an RPC marker. h. Relative proportions of major cell classes across different datasets analyzed in panel e separated by age. T, this study.

Article Snippet: Clumps were removed using a 40 μ m cell strainer and the cell suspension was spun down and re-suspended in Ames + 4% BSA at a concentration of 10 million cells per 100 μ l. Cells from E13, E14, E16, and P0 were incubated for 15 minutes at room temperature with antibodies to Thy1 (also known as CD90) and L1CAM pre-conjugated to the fluorophores APC (ThermoFisher Scientific#17-0902-82) and PE (Miltenyi Biotec 130-102-243), respectively.

Techniques: Staining, Marker, Whisker Assay, Gene Expression, Expressing

Journal: bioRxiv

Article Title: Diversification of multipotential postmitotic mouse retinal ganglion cell precursors into discrete types

doi: 10.1101/2021.10.21.465277

Figure Lengend Snippet: a-c. UMAP embedding for RGCs at E14 (a, same as ) , E16 (b, same as ) and P0 (c, same as ) with cells colored by enrichment method showing comparable transcriptomic diversity of immature RGCs enriched by L1cam or Thy1. d. Simpson and Shannon diversity indices (see Methods ) associated with clustering decrease and increase with age respectively, consistent with increasing transcriptomic diversity.

Article Snippet: Clumps were removed using a 40 μ m cell strainer and the cell suspension was spun down and re-suspended in Ames + 4% BSA at a concentration of 10 million cells per 100 μ l. Cells from E13, E14, E16, and P0 were incubated for 15 minutes at room temperature with antibodies to Thy1 (also known as CD90) and L1CAM pre-conjugated to the fluorophores APC (ThermoFisher Scientific#17-0902-82) and PE (Miltenyi Biotec 130-102-243), respectively.

Techniques:

Journal: Open Medicine

Article Title: CHL1 and NrCAM are Primarily Expressed in Low Grade Pediatric Neuroblastoma

doi: 10.1515/med-2019-0109

Figure Lengend Snippet: Expression of CHL1 and NrCAM in pediatric neuroblastoma. Representative examples of CHL1 positive (A) and CHL-1 negative (B) (magnification x100) as well as NrCAM positive (C) and NrCAM negative (D) immunostaining (magnification x200).

Article Snippet: Afterwards, the primary antibody either specific for CHL1 (goat, polyclonal antibody: AF2126, R&D Systems, MN, USA) or NrCAM (goat anti-human NrCAM antibody: AF2034, R&D Systems, MN, USA,) was applied at 37°C and pH 9.1 for 60 minutes.

Techniques: Expressing, Immunostaining

Journal: Open Medicine

Article Title: CHL1 and NrCAM are Primarily Expressed in Low Grade Pediatric Neuroblastoma

doi: 10.1515/med-2019-0109

Figure Lengend Snippet: Kaplan-Meier survival curves for overall and event-free survival. No association was found for CHL1-expression (A/B). Survival rates were better by trend in children with NrCAM positive tumors (C/D) but without statistical significance (p=0.07 and p=0.06).

Article Snippet: Afterwards, the primary antibody either specific for CHL1 (goat, polyclonal antibody: AF2126, R&D Systems, MN, USA) or NrCAM (goat anti-human NrCAM antibody: AF2034, R&D Systems, MN, USA,) was applied at 37°C and pH 9.1 for 60 minutes.

Techniques: Expressing

Journal: Open Medicine

Article Title: CHL1 and NrCAM are Primarily Expressed in Low Grade Pediatric Neuroblastoma

doi: 10.1515/med-2019-0109

Figure Lengend Snippet: CHL1 expression as well as clinical, pathologic and molecular characteristics of the analysed neuroblastoma tissue samples. Statistical analyses by using cross-tables, two-sided Fisher´s and Chi-squared test.

Article Snippet: Afterwards, the primary antibody either specific for CHL1 (goat, polyclonal antibody: AF2126, R&D Systems, MN, USA) or NrCAM (goat anti-human NrCAM antibody: AF2034, R&D Systems, MN, USA,) was applied at 37°C and pH 9.1 for 60 minutes.

Techniques: Expressing, Amplification