Journal: bioRxiv

Article Title: Optimal AAV capsid/promoter combinations to target specific cell types in the common marmoset cerebral cortex

doi: 10.1101/2024.05.09.593444

Figure Lengend Snippet: ( A ) Scheme to identify EGFP-expressing cell types by fIHC. Neurons and astrocytes were immunolabeled for NeuN and GFAP, respectively. Cells immunostained for Olig2 or Iba1 were identified as oligodendrocytes and microglia. Cells were detected with Hoechest 33342, NucBlue, in the mounting reagent ProLong Glass. ( B ) Representative immunohistochemical images of marmoset cerebral cortex that received injection of AAV2. Two serial slices were presented: one immunolabeled for GFP, NeuN, and GFAP, and another one for GFP, Olig2, and Iba1, as indicated at each panel. Scale bar, 100 μm.

Article Snippet: Tissue sections were reacted overnight at room temperature by immersion in following primary antibodies in blocking solution (2% Donkey Serum (S30-100ML, Merck), BSA (01862-87, Nacalai Tesque, Kyoto, Japan), 0.5% Triton X-100, 0.03% NaN3 in 1 x PB): rat monoclonal anti-GFP antibody (1:1,000; 04404-84; Nacalai Tesque, Kyoto, Japan), mouse monoclonal anti-NeuN antibody (1:1,000; MAB377; Merck), rabbit polyclonal anti-GFAP antibody (1:200; GFAP-Rb-Af800; Nittobo Medical, Tokyo, Japan), rabbit polyclonal anti-S-100β antibody (1:200; S100b-Rb-Af1000, Nittobo Medical), mouse monoclonal anti-Olig2 antibody (1:500; MABN50; Merck) and rabbit polyclonal anti-Iba1 antibody (1:500; 019-19741; Fujifilm Wako Chemicals, Tokyo, Japan).

Techniques: Expressing, Immunolabeling, Immunohistochemical staining, Injection

Journal: bioRxiv

Article Title: Optimal AAV capsid/promoter combinations to target specific cell types in the common marmoset cerebral cortex

doi: 10.1101/2024.05.09.593444

Figure Lengend Snippet: ( A ) Schema depicting the AAV genome structure. ( B - C ) Immunolabeled fluorescent images of EGFP in the cerebral cortex that received injection of AAV5 ( B ) or AAVrh10 ( C ) vectors expressing EGFP by the mMBP promoter. The middle immunofluorescence images present an overlay of immunolabeling for EGFP and the oligodendrocyte marker Olig2. The bottom images are magnifications of the boxed areas in the center images. Scale bar, 100 μm. ( D - E ) Summary graphs showing the specificity ( D ) and efficiency ( E ) of oligodendrocyte transduction. The dotted line in the graph indicates a ratio of oligodendrocytes to total cells present in the marmoset cortex (Figure S3). Error bars indicate S.E.M., and dots in the graph indicate the respective values for each of the individual marmosets. Asterisks indicate statistically significant differences between the AAV5 and AAVrh10. ** p < 0.01, *** p < 0.001 by student’s t-test.

Article Snippet: Tissue sections were reacted overnight at room temperature by immersion in following primary antibodies in blocking solution (2% Donkey Serum (S30-100ML, Merck), BSA (01862-87, Nacalai Tesque, Kyoto, Japan), 0.5% Triton X-100, 0.03% NaN3 in 1 x PB): rat monoclonal anti-GFP antibody (1:1,000; 04404-84; Nacalai Tesque, Kyoto, Japan), mouse monoclonal anti-NeuN antibody (1:1,000; MAB377; Merck), rabbit polyclonal anti-GFAP antibody (1:200; GFAP-Rb-Af800; Nittobo Medical, Tokyo, Japan), rabbit polyclonal anti-S-100β antibody (1:200; S100b-Rb-Af1000, Nittobo Medical), mouse monoclonal anti-Olig2 antibody (1:500; MABN50; Merck) and rabbit polyclonal anti-Iba1 antibody (1:500; 019-19741; Fujifilm Wako Chemicals, Tokyo, Japan).

Techniques: Immunolabeling, Injection, Expressing, Immunofluorescence, Marker, Transduction

Journal: Frontiers in Pharmacology

Article Title: Clemastine Ameliorates Perioperative Neurocognitive Disorder in Aged Mice Caused by Anesthesia and Surgery

doi: 10.3389/fphar.2021.738590

Figure Lengend Snippet: Effects of clemastine on expression levels of OLIG2 and MBP in hippocampus of aged mice after anesthesia and surgery. ( A ) Relative mRNA expressions of OLIG2 and MBP, normalized to that of the GAPDH internal control. ( B ) Representative western blot images of OLIG2 and MBP. ( C ) Relative protein expressions of OLIG2 and MBP, normalized to that of the β-tubulin internal control. The data are presented as mean ± S.D. ( n = 10 mice per group). * p < 0.05 compared with the CON group. **** p < 0.001 compared with the CON group. ### p < 0.005 compared with the PND group. #### p < 0.001 compared with the PND group.

Article Snippet: Rabbit polyclonal anti-mouse TNF-α antibody (1:1,000, AF8208, Beyotime, CHN), rabbit polyclonal anti-mouse IL-1β antibody (1:1,000, AF7209, Beyotime, CHN), rabbit polyclonal anti-mouse WNT10B antibody (1:1,000, DF9038, Affinity, CHN), rabbit polyclonal anti-mouse β-catenin antibody (1:1,000, AF5126, Beyotime, CHN), rabbit monoclonal anti-mouse OLIG2 antibody (1:1,000, AF1312, Beyotime, CHN), rabbit polyclonal anti-mouse MBP antibody (1:1,000, BA0094, Boster, CHN), rabbit polyclonal anti-mouse SNAP25 antibody (1:1,000, AF8016, Beyotime, CHN), rabbit monoclonal anti-mouse PSD95 antibody (1:1,000, AF1096, Beyotime, CHN), rabbit monoclonal anti-mouse BDNF antibody (1:1,000, AF1423, Beyotime, CHN) and rabbit monoclonal anti-mouse β-tubulin antibody (1:1,500, AF1216, Beyotime, CHN) were the primary antibodies used.

Techniques: Expressing, Control, Western Blot

Journal: Regenerative Biomaterials

Article Title: Alginate hydrogel cross-linked by Ca 2+ to promote spinal cord neural stem/progenitor cell differentiation and functional recovery after a spinal cord injuryhh

doi: 10.1093/rb/rbac057

Figure Lengend Snippet: Fate of NSPCs within AH channels post-transplantation. ( A–D ) GFP-labeled NSPCs (green) survived within and around the AH scaffold post-implantation. ( E ) Quantification of GFP (+) cells within the channels at different time points. ( F–H ) NSPCs within the channels differentiated into mature neurons labeled for NeuN (red). ( I ) Quantification of differentiated neurons (GFP/NeuN double-labeled) at different time points. ( J – L ) NSPCs (green) in the channels differentiated into astrocytes were identified by GFAP labeling (red) at 8 weeks post-transplantation. ( M ) Quantification of differentiated astrocytes (GFP/GFAP double-labeled) at different time points. ( N–P ) Grafted NSPCs also differentiated into oligodendrocytes labeled for Olig2 (red) at 8 weeks post-transplantation. ( N ) Quantification of differentiated oligodendrocytes (GFP/Olig2 double-labeled) at different time points (* P < 0.05; ** P < 0.01; *** P < 0.001; one-way ANOVA followed by Tukey’s post hoc analysis). Scale bar: (A–D) 500 μm and (F–H, J–L and N–P) 50 μm.

Article Snippet: Samples were incubated with the following primary antibodies: rabbit anti-GFP (1:2000; ABclonal), mouse anti-Tuj-1 (1:2000; Santa Cruz), mouse anti-glial fibrillary acidic protein (GFAP, 1:1500; ABclonal), mouse anti-nestin (1:1000; Santa Cruz) and mouse anti-Olig2 (1:500; ABclonal).

Techniques: Transplantation Assay, Labeling

Journal: Regenerative Biomaterials

Article Title: Alginate hydrogel cross-linked by Ca 2+ to promote spinal cord neural stem/progenitor cell differentiation and functional recovery after a spinal cord injuryhh

doi: 10.1093/rb/rbac057

Figure Lengend Snippet: Fate of NSPCs in the lesion and effects of NSPCs on axonal regrowth in NSPC-grafted animals without AHs. ( A ) Comparison of NSPC survival in hydrogels/lesion site between the NSPC-only and AHs-NSPC groups at 8 weeks post-engraftment (*** P < 0.001, unpaired Student’s t -test). The percentage of surviving cells in the NSPC group was calculated as the proportion of the total number of grafted cells (1.25 × 10 5 cells). ( B ) Comparison of cell fates between the NSPC-only and AHs-NSPC groups for neurons, astrocytes and oligodendrocytes (n.s., P > 0.05, * P < 0.05, unpaired Student’s t -test). ( C ) Grafted NSPCs (green) differentiated into neurons (co-labeled forTuj-1) and supported host axonal regeneration (red only) in the lesion. ( C 1 and C 2 ) Higher magnifications of the boxed area in ( C ) shows axons sprouting from grafted NSPCs (arrowheads) and host neurons (arrows and area marked with a star) arranged in a randomized pattern. ( D ) NSPCs (green) partly filled the lesion and differentiated into astrocytes (co-labeled for GFAP). Cavitations associated with atrophic stumps were observed around the lesion. ( D 1 ) is a higher magnification of the boxed area in ( D ), and ( D 2 ) is a higher magnification of the boxed area in ( D 1 ) to better visualize the differentiated astrocytes. NSPCs (green) differentiated into oligodendrocytes (co-labeled for Olig2). ( E 1 ) is a higher magnification of the boxed area in ( E ), and ( E 2 ) is a higher magnification of the boxed area in (E 1 ) to show differentiated oligodendrocytes. Scale bar: (C–E) 500 μm; (C 1 , C 2 , D 1 and E 1 ) 100 μm; and (D 2 and E 2 ) 20 μm.

Article Snippet: Samples were incubated with the following primary antibodies: rabbit anti-GFP (1:2000; ABclonal), mouse anti-Tuj-1 (1:2000; Santa Cruz), mouse anti-glial fibrillary acidic protein (GFAP, 1:1500; ABclonal), mouse anti-nestin (1:1000; Santa Cruz) and mouse anti-Olig2 (1:500; ABclonal).

Techniques: Labeling