isl1 ms dshb 39 4d5  (Developmental Studies Hybridoma Bank)

Journal: PLOS ONE

Article Title: Human iPSC-derived myelinating organoids and globoid cells to study Krabbe disease

doi: 10.1371/journal.pone.0314858

Figure Lengend Snippet: Antibodies.

Article Snippet: Islet1/2 , DSHB 39.4D5 , AB_2314683 , IHC 1:100.

Techniques:

Journal: bioRxiv

Article Title: Laminin β4 is required for the development of human peripheral sensory neurons

doi: 10.1101/2024.11.22.624899

Figure Lengend Snippet: A) Effects of LAMB4 downregulation in SNs. Brightfield images of SNs differentiated from LAMB4 +/+ , LAMB4 +/− , and LAMB4 −/− hPSCs at indicated days. B) Expression of SN markers upon loss of LAMB4 . LAMB4 +/+ , LAMB4 +/− , and LAMB4 −/− SNs were fixed on day 20 and stained for peripheral neuron markers (TUJ1 and PRPH) and SN markers (BRN3A, ISL1). Nuclei were stained with DAPI. C) Percentage of BRN3A + cells in B) . Normalized to DAPI. D) Percentage of ISL1 + cells in B) . Normalized to DAPI. E) Quantification of the number of SNs differentiated from LAMB4 mutant hPSCs. SNs from LAMB4 +/+ , LAMB4 +/− , and LAMB4 −/− cells were harvested on day 20. SNs were then fixed, stained for BRN3A, and analyzed by flow cytometry (n=4 biological replicates). F) Expression of SN markers in LAMB4 mutant SNs. RNA from LAMB4 +/+ , LAMB4 +/− , and LAMB4 −/− SNs was isolated on day 20 and gene expression was measured by RT-qPCR (n=5 biological replicates). G) Electrical activity of SNs upon loss of LAMB4 . The firing rate of LAMB4 +/+ , LAMB4 +/− , and LAMB4 −/− SNs was measured using multi-electrode array (MEA). Each dot represents the mean firing rate of 6 wells measured over 40 days (n=4 biological replicates). H) Electrophysiological changes of SNs to pharmacological nociceptor or mechanoreceptor activators. LAMB4 -mutant SNs were incubated with nociceptor agonists (0.25 µM capsaicin and 1 µM WIN55,212-2) and a mechanoreceptor activator (hypoosmotic medium) and the electrical activity was measured using MEA. Each dot represents the mean firing rate of 6 wells measured over 40 days (n=4 biological replicates). I) Degeneration of LAMB4 mutant SNs. LAMB4 +/+ , LAMB4 +/− , and LAMB4 −/− SNs were cultured in plates coated with fibronectin and poly-L-ornithine and reduced NGF concentration (1 ng/mL) and fixed on the indicated days. Cells were then stained for the neuronal marker TUJ1, the SN marker BRN3A, and DAPI. J) Quantification of BRN3A + SNs from I) (n=3-4 biological replicates). For C) , D) , E) , and G) one-way ANOVA followed by Tukey’s multiple comparisons test. For F) and H) , one-way ANOVA followed by Dunnett’s multiple comparisons test. For J) , two-way ANOVA followed by Šídák’s multiple comparisons test. ns, non-significant, *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. Graphs show mean ± SEM.

Article Snippet: Laminin β4 (Abcam, cat# ab150819; Sigma, cat# HPA020242), laminin β1 (Abcam, cat# ab44941), laminin α4 (R&D Systems, cat# AF7340), laminin γ3 (Proteintech, cat# 67261-1-I), SOX10 (Santa Cruz, cat# sc-365692), TFAP2A (Abcam, cat# ab108311), BRN3A (Millipore, cat# MAB1585), TUJ1 (Biolegend, cat# 801201), ISL1 (DSHB, cat# 39.4D5-c), PRPH (Santa Cruz, cat# sc-377093), Actin (BD Biosciences, cat# 612656), Vinculin (Abclonal, cat# A14193), αSMA (Sigma, cat# A5228), Phalloidin-iFluor 488 (Abcam, cat# ab176753), CD49d-PE/Cy7 (Biolegend, cat# 304314), TRKA-PE (R&D Systems, cat# FAB1751P), TRKB-AF647 (R&D Systems, cat# FAB3971R), and TRKC-PE (R&D Systems, cat# FAB373P).

Techniques: Expressing, Staining, Mutagenesis, Flow Cytometry, Isolation, Quantitative RT-PCR, Activity Assay, Incubation, Cell Culture, Concentration Assay, Marker