Fish skin gelatin

Paraffin sections were dewaxed and rehydrated as described above. Antigen retrieval was performed in 10 mM sodium citrate, pH 6.0, in an autoclave for 40 min at 121°C. Samples were cooled to ambient temperature and equilibrated in 1× TBS (50 mM TBS, pH 7.4) for 5 min, followed by blocking in 4% nonimmune goat serum, 0.1% cold water fish skin gelatin (Sigma-Aldrich), and 0.1% Triton X-100, in 1× TBST (150 mM NaCl, 1% Tween 20, and 50 mM Tris, pH 7.5) for 30 min. Sections were washed in 1× TBST and incubated with primary antibodies or 488-conjugated Lotus tetragonolobus agglutinin (Vector Laboratories) diluted in 0.1% cold water fish skin gelatin in 1× TBST overnight at 4°C. Next, samples were washed with 1× TBST and incubated with 1:1,000 dilutions of secondary antibodies diluted in 0.1% cold water fish skin gelatin in 1× TBST for 1 h. Last, the sections were washed with 1× TBST, rinsed with 1× TBS, dipped in 1× TBS supplemented with 1 mg/ml DAPI for 5 s, and mounted with Prolong Gold (Thermo Fisher Scientific; San Agustin et al., 2016 (link)).