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 ) Fluorescent images of the cortex immunolabeled for EGFP 4 weeks after AAV injection. Scale bar, 100 μm. (B-E) Graphs showing the ratio of GFP (+) neurons ( B ), GFP (+) oligodendrocytes ( C ), GFP (+) astrocytes ( D ), and GFP (+) microglia (E) to total GFP (+) cells. The dotted lines in the graphs are ratios of respective cell types to total cells present in the marmoset cortex (Figure S3). Error bars indicate S.E.M., and the dots in the graph indicate the respective values for each of the individual marmosets. The asterisks indicate a statistically significant difference between the AAV5 vector and the other capsids. * p < 0.05, ** p < 0.01 by 1-way ANOVA with Tukey’s post hoc test. n.s., not statistically significant.

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, Plasmid Preparation

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 ) Immunofluorescent EGFP images of the cortex injected with AAV7 expressing EGFP by the CBh, CMV, or CAG promoter. Scale bar, 100 μm. ( B - E ) Graphs showing ratios of respective EGFP-immunolabeled cell types to total EGFP-expressing cells by AAV7 vectors with the CBh, CMV, and CAG promoters. The dotted lines in the graphs are ratios of respective cell types 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. P values obtained using 1-way ANOVA with Tukey’s post hoc test were described in the graphs. n.d., not detected.

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: Injection, Expressing, Immunolabeling

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 ) Immunofluorescent EGFP images of the cerebral cortex received injections of respective AAV vectors. Scale bar, 100 μm. ( C ) Representative image immunostained for EGFP alone (left) and merged image for EGFP and GFAP (right) after injection of the AAV2 vector. Scale bar, 100 μm. ( D ) Graph showing the percentage of GFAP-positive astrocytes to total EGFP-expressing cells 4 weeks after injection of the AAV vector as indicated. The dotted line in the graph shows 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. n.s., not statistically significant by one-way ANOVA with Tukey’s post hoc 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: Injection, Plasmid Preparation, Expressing

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