Immunostaining of Mouse Craniofacial Tissues

Samenvatting

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 staining dish with a steamer or water bath to 95–100 °C. Immerse the slides in the citrate buffer and incubate for 10 min.
   2. Take the staining dish out of the steamer or water bath and cool the slides at room temperature for 20 minutes or longer15.
      NOTE: As alternatives, use Tris-EDTA buffer (10 mM Tris base, 1mM EDTA, 0.05% Tween 20, pH 9.0) or EDTA buffer (1 mM EDTA, 0.05% Tween 20, pH 8.0) for heat-induced antigen retrieval. Use a pressure cooker, microwave, or water bath for heat-induced antigen retrieval in addition to the hot steamer. An enzyme-induced antigen retrieval using trypsin or pepsin is another alternative. Optimize the concentration and treatment time of enzymatic retrieval to avoid damaging sections. Optimize the antigen retrieval method for each antibody/antigen combination.
3. Incubate each slide with 200 μL of blocking solution (5% donkey serum diluted in 0.1% PBST) at RT for 30 min, then remove the blocking solution without rinse.
4. Incubate each slide with 100 μL of primary antibody or antibodies diluted in blocking solution for 1 h at RT or O/N at 4 °C. Rinse slides with PBS three times for 10 min each at RT.
5. Incubate each slide with 100 μL of secondary antibody diluted in blocking solution for 1 h at RT. Rinse slides in PBS three times for 10 min each at RT. Protect slides from light.
6. Mount slides.
   1. Add two drops of anti-fade medium with DAPI (4', 6-diamidino-2-phenylindole) on the slide. Then cover with a coverslip.
   2. Store at 4 °C in the dark until ready to image.
      NOTE: As an alternative, label the nuclei with DAPI or Hoechst 33324 dye diluted 1:2,000 in PBS at RT first, then mount with glycerol.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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