Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone

Summary

Here, we present a detailed protocol to detect and quantify protein levels during craniofacial morphogenesis/pathogenesis by immunostaining using mouse craniofacial tissues as examples. In addition, we describe a method for preparation and cryosectioning of undecalcified hard tissues from young mice for immunostaining.

Abstract

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and simple protocol to detect protein expression during craniofacial morphogenesis/pathogenesis using mouse craniofacial tissues as examples. The protocol consists of preparation and cryosectioning of tissues, indirect immunofluorescence, image acquisition, and quantification. In addition, a method for preparation and cryosectioning of undecalcified hard tissues for immunostaining is described, using craniofacial tissues and long bones as examples. Those methods are key to determine the protein expression and morphological/anatomical changes in various tissues during craniofacial morphogenesis/pathogenesis. They are also applicable to other tissues with appropriate modifications. Knowledge of the histology and high quality of sections are critical to draw scientific conclusions from experimental outcomes. Potential limitations of this methodology include but are not limited to specificity of antibodies and difficulties of quantification, which are also discussed here.

Introduction

The face is a key part of human identity, and is composed of several different types of tissues, such as epithelium, muscle, bone, cartilage, tooth. Those tissues are derived from all three germ layers: ectoderm, endoderm, and mesoderm^1,2. For proper patterning and development of craniofacial tissues, cell proliferation, death and differentiation need to be highly coordinated and regulated by specific signaling pathways, such as Wnt, Fgf, Hh and Bmp pathways^3,4,5. Defects in proliferation, survival or differentiation ....

Protocol

All mouse experiments were carried out in accordance with University of Michigan guidelines covering the humane care and use of animals in research. All animal procedures used in this study were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Michigan (Protocol #PRO00007715).

1. Tissue Preparation

1. Preparation of embryonic tissues
   1. Prepare one 10 cm dish and several 3.5 cm dishes containing phosphate buffered saline (PBS), and one 12-well culture plate containing 2 mL 4% paraformaldehyde (PFA) in PBS in each well for each pregnant mouse. Place all Petri dishes ....

Results

Embryonic craniofacial tissue sections
Following the above steps, heads were dissected from control (P0-Cre) or mutant (constitutively activated Bmpr1a in neural crest cells, P0-Cre; caBmpr1a) embryos at embryonic day (E) 16.5 or 18.5. After fixing in 4% PFA for 4 h, samples were embedded in OCT and cryosectioned coronally. Resulted sections were immunostained with antibodies against pSmad1/5/9 (downstream BMP signaling factors) or Ki67 (a cell proli.......

Discussion

Here we provide a detailed protocol for preparation of mouse head and undecalcified bone tissues, and cryosectioning for immunostaining of cell proliferation, cell death, and BMP signaling markers. We also detail the strategy for obtaining quantitative data from immunofluorescent images. Those methods can also be applicable to other tissues with appropriate modifications.

Conditions for tissue preparation vary by the size and type of tissues. The fixation and cryoprotection time usually need s.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Adhesive tape	Leica	#39475214
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
Gelatin	Sigma	G1890
In Situ Cell Death Detection Kit	Millipore	S7165
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
Proteinase K	Invitrogen	AM2542
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
Sucrose	Sigma	S9378
Tris	Sigma	10708976001