Vector trueview quenching kit

For IF staining, the procedure was similar to IHC, except that the endogenous peroxidase blocking step was omitted and fluorochrome-conjugated secondary antibodies were used. To reduce autofluorescence, sections were incubated in Vector TrueView quenching kit (Vector Lab, USA) for 2–5 min. Finally, sections were mounted in Fluoromount (Sigma) and examined using fluorescence microscope.

Prior to immunofluorescence staining, 5 µm thick paraffin sections of sheep explants were dewaxed and rehydrated to water. Antigen retrieval was carried out by microwaving the slides in 0.1 M citrate buffer. To reduce non-specific binding, the slides were incubated with 10% normal goat serum in 2% bovine serum albumin (BSA -Sigma) for 40 min. Specimens were then incubated overnight in a moist chamber with a cocktail of antibodies consisting smooth muscle alpha actin (mouse from DAKO) and CD31 (rabbit- Abcam) Negative controls consisted of 10% normal goat serum in 2% BSA in PBS. After thorough washing, all the specimens were incubated with secondary antibodies, goat anti rabbit Alexa Fluor 634 for rabbit polyclonals and goat anti mouse Alexa Fluor 488 (Life Technologies) for 1 h at 1:1000 dilution. After washing several times with PBS sections were incubated with DAPI (1:5000) (sigma) for 15 min and to reduce autofluorecence, sections were incubated with Vector TrueView quenching kit (Vector laboratories) for 3 min and mounted using Permafluor (Beckman Coulter). Stained sections were imaged using a Zeiss LSM 880 confocal microscope.