Affinipure goat anti mouse igg igm fab h l

Frozen sections were left at room temperature for 20 min, and then incubated in phosphate buffered saline-Tween 20 (PBST) solution for 5 min. Sections were treated with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) for 10 min and rinsed with PBST for 5 min. Sections were microwaved in sodium citrate for 20 min. After 20 min siting at room temperature, sections were rinsed with PBST for 5 min. Sections were blocked with 3% bovine serum albumin (BSA) and 10% AffiniPure Goat Anti-Mouse IgG + IgM Fab (H+L) (Jackson ImmunoResearch, 115-005-044) PBST solution for 1 h at room temperature. After washed with PBST for 5 min, sections were incubated with primary antibodies diluted in 3% BSA and 5% Donkey Serum PBST solution at 4 °C for overnight. Sections were then washed with PBST for 3 times, 5 min each, and then incubated with secondary antibodies conjugated with fluorescent dye Alexa 594 or Alexa 488 diluted in 3% BSA PBST (1:400) for 1 hour at room temperature. Sections were then incubated in PBST containing Hoechst (1:15 000) for 5 min, followed by wash with PBST 2 times, 5 min each. The sections were mounted with coverslips using Fluomount-G solution (Southern Biotech, 0100–01). Immunostaining images were taken using a confocal microscope (Olympus FluoView 500, Center Valley, PA).