Whole-Mount Indirect Fluorescent Immunohistochemistry – Double or multiple staining (Version 1.0)

Version

1.0

Notice

This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.

Authors

Kevin Gaido

Keywords

[‘immunofluorescence’]

Subjects

[‘Cell biology’, ‘Developmental biology’, ‘Gene expression analysis’]

Release Date

2017-08-07

Abstract

Whole-Mount Indirect Fluorescent Immunohistochemistry – Double or multiple staining protocol by the Gaido Group.

Procedure

• All incubations and washes are carried out at 4 ºC on an orbital shaker.

1. Tissue samples are fixed with 4% paraformaldehyde in PBS (1:4, purchased 16%, diluted with PBS), overnight, 4 ºC. (Keep samples in tubes?)
2. Rinse, 2 times in PBS.
3. Quench in 50 mM NH4CL, 15 min.
4. Block in blocking buffer, 24 hr (1% BSA, 0.1% Saponin, 0.02% sodium azide in PBS) [100mg BSA+10ul Saponin+2ul sodium azide ——à10 ml PBS]. You also need to try blocking with Triton X-100 (0.1%) instead of saponin.
5. Incubated in FIRST primary antibody in fresh blocking buffer, 24 hr, 4 ºC. You can incubate all primary antibodies at the same time.
6. Wash 5 times, 1 hr/each, in blocking buffer.
7. Incubated in secondary antibody in fresh blocking buffer, 24 hr, 4 ºC. You can incubate all secondary antibodies at the same time.
8. Wash 5 times, 1 hr/each, in blocking buffer.

Fluorescently labeled samples are image by laser scanning confocal microscope. Whole-mount samples are viewed in antifade buffer with glycerol in a culture dish with attached coverslip. For viewing, use the special coverslip glass-bottomed culture dishes without a coverslip.

Consortium

GenitoUrinary Development Molecular Anatomy Project (GUDMAP) Consortium