Donkey anti rabbit fab fragment

Paraffin sections of colon and skin were deparaffinized for 5 min each with: xylene twice, 100% ethanol twice, 95% ethanol twice, 75% ethanol, ddH2O. Antigen retrieval was performed with a Decloaking Chamber (NxGen) using a buffer containing 1.27 mM EDTA, 3.75 mM boric acid, 0.61 mM sodium borate and 0.003% ProClin. The slides were then blocked for 1 h (23 °C) with 5% BSA and 15% donkey serum in PBS. ARPC1B antibody (Sigma-Aldrich, HPA004832; dilution 1/100 in blocking buffer) kept overnight at 4 °C in a humidified staining chamber. After washing the slides three times with PBS containing 0.05% Tween 20, they were stained with Alexa Fluor 594 donkey anti-rabbit Fab fragment (Jackson ImmunoResearch; dilution 1/250) for 1 h at (23 °C). Slides were blocked with donkey anti-rabbit Fab fragment (Jackson ImmunoResearch; dilution 1/100) for 1 h (23 °C), then incubated with polyclonal rabbit anti-ARPC1A (Sigma-Aldrich, HPA004334; dilution 1/25) overnight at 4 °C. Secondary Alexa Fluor 488 donkey anti-rabbit (Jackson ImmunoResearch; dilution 1/250) was added for 1 h at RT. To minimize autofluorescence the slides were treated with 3.3 mM Sudan Black in 70% ethanol for 10 min (23 °C). Slides were then stained with DAPI (dilution 1/5,000) and mounted with Dako fluorescent mounting medium (S3023).

For single fluorescent labeling, samples were blocked with 5% normal serum and incubated overnight at 4°C with rabbit anti–h-αS (1:3000). For double fluorescence, sections were first incubated overnight with either rabbit anti-RFP (1:3000) or mouse anti–Syn-O2 (1:2000; TAB-0748CLV, Creative Biolabs) and then incubated with anti–h-αS. Labeling of these primary antibodies was achieved using a secondary antibody conjugated with DyLight 488 or DyLight 594 (1:300; Vector Laboratories). Sections were rinsed, mounted on coated slides, and coverslipped with Vectamount mounting medium (Vector Laboratories). For triple fluorescence, tissue sections were processed using the following sequential labeling procedures. First, for detection of mCherry-conjugated hM3D, mCherry was labeled by incubations with rabbit anti-RFP (1:3000), donkey anti-rabbit Fab fragment (1:200; Jackson ImmunoResearch), and goat anti-donkey Alexa Fluor 594 (1:300; Abcam). Second, SOD2 was labeled by incubations with rabbit anti-SOD2 (1:1000) and goat anti-rabbit Alexa Fluor 647 (1:300, Abcam). Last, h-αS was labeled by overnight incubation with rabbit anti–h-αS conjugated with Alexa Fluor 488 (1:400; ab216124, Abcam). Fluorescence images were collected on Zeiss microscopes (LSM700, LSM800, LSM880, or LSM900) using the ZEN software (Carl Zeiss).