Immunostaining of Mouse Craniofacial Tissues

Abstract

Source: Yang, J., et al., Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone. J. Vis. Exp. (2019)

This video demonstrates the immunofluorescence staining of cryosections from mouse embryonic craniofacial tissues, using fluorescence microscopy to assess nuclear marker expression and determine cell proliferation and signaling activity.

Protocol

Disclaimer text: All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.    

1. Immunofluorescence staining

1. Take out slides from -80 °C. Keep slides at RT for 1 h to airdry sections. Rinse slides in 0.1% PBST (0.1% Polyethylene glycol tert-octylphenyl ether in PBS; see Table of Materials) three times for 5 min each to wash out OCT and permeabilize sections.
2. Optionally, perform antigen retrieval (optional).
   1. Preheat the citrate buffer (10 mM sodium citrate pH 6) in the sta....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alexa fluor 488-goat anti-Rabbit secondary antibody	Invitrogen	A-11034
Antifade Mountant with DAPI	Invitrogen	P36931
Bovine serum albumin	Sigma	A2153
Coverslips	Fisher Brand	12-545-E
Cryostat	Leica	CM1850
EDTA	Sigma	E6758
Fluorescence microscope	Olympus	BX51
Microscope slides	Fisher Brand	12-550-15
OCT Compound	Fisher Healthcare	23-730-571
Paraformaldehyde (PFA)	Sigma	P6148
Phosphate buffered saline (PBS)	Sigma	P4417
Polyethylene glycol tert-octylphenyl ether	Sigma	T9284	Triton X-100
Rabbit anti-Ki67 antibody	Cell Signaling Technology	9129	Lot#:3; RRID:AB_2687446
Rabbit anti-pSmad1/5/9 antibody	Cell Signaling Technology	13820	Lot#:3; RRID:AB_2493181
Sodium citrate	Sigma	1613859
Tris	Sigma	10708976001