Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone

Here, we present our simple protocol for immunofluorescence detection on proteins on sectioned materials. This method does not require antigen retrieval. We will use mouse heads, including undecalcified hard tissues.

The main advantage of this technique is to skip antigen retrieval and decalcification of hard tissues. Those lead to a good quality of immunostaining results. This method also saves both time and labor.

This method is also applicable to other tissues with appropriate modifications. To make nice sections from undecalcified tissues, dissect your target tissue further to trim surrounding tissues, and if necessary treat the samples with 10%EDTA at four degrees Celsius for two days before cryoprotection. Visual demonstration provides a detailed and a clear explanation of the procedure of this method.

To begin, prepare one 10-centimeter and several 3.5-centimeter dishes containing PBS and one 12-well culture plate containing two milliliters of 4%PFA in PBS in each well for each pregnant mouse, and place them all on ice. To dissect embryos from pregnant mice, grab the skin below the center of the belly of a euthanized mouse with forceps, cut through the skin only, and then gently pull at the skin to separate it from the underlying abdominal muscle wall. Following the same line of the skin incision, cut into the abdominal cavity.

Remove the uterus containing a string of embryos. Then, remove the embryos by gently cutting away the uterine wall, which will remove the extraembryonic tissues such as the yolk sac and amnion. Cut and isolate the head from each embryo, and with forceps transfer each head into a separate well of a 12-well plate containing 4%PFA.

Incubate at four degrees Celsius for four hours to fix. Rinse the heads in PBS at four degrees Celsius with gentle shaking for 12 hours. To cryoprotect the heads, transfer them using forceps into a new 12-well plate containing two milliliters of 30%sucrose in PBS.

Incubate with gentle agitation at four degrees Celsius until the heads sink to the bottom of the dish. To embed the cryoprotected heads, transfer them into a mold containing Optimal Cutting Temperature Compound, and equilibrate for several minutes. Using forceps, adjust the location and direction of the samples so that the trimmed side of the samples faces the bottom of the embedding mold.

Then, place the mold on dry ice to freeze, and store in a plastic bag at minus 80 degrees Celsius until ready for cryosectioning. For postnatal tissue, after removing the skin and adipose tissues of a euthanized, three-weeks to three-months-old mouse, cut and isolate the head and remove the mandible. Then, fix and cryoprotect the head as done with embryo heads.

Embed in 8%gelatin in a similar manner as embryo heads in OCT, and keep the Cryomolds in a plastic bag at minus 80 degrees Celsius until cryosectioning. Set the appropriate cryostat temperature. Place the samples in the cryostat chamber for about 30 minutes to equilibrate to the cryostat temperature.

Remove the block with the sample from the Cryomold, and mount it with an OCT drop onto the specimen chuck and freeze it, making sure to keep the trimmed side of the sample furthest from the chuck and facing the operator. Load the block-mounted chuck onto the cryostat object holder. Adjust the blade holder so that the blade angle is three to five degrees relative to the sample.

Collect 10-micrometer sections onto coated microscope slides. Leave the sections at room temperature until completely dry, and then store them at minus 80 degrees Celsius. To begin with the staining, take out slides from minus 80 degrees Celsius, and keep them at room temperature for one hour to air-dry the sections.

Rinse the slides in 0.1%PBST three times for five minutes each to wash out OCT and permeabilize the sections. Add 200 microliters of blocking solution to each slide, and incubate at room temperature for 30 minutes. Then, remove the blocking solution without rinsing the slide.

Add 100 microliters of primary antibody diluted in blocking solution to each slide, and incubate overnight at four degrees Celsius. Rinse the slides with PBS three times for 10 minutes each at room temperature. Add 100 microliters of secondary antibody diluted in blocking solution, and incubate for one hour at room temperature, protected from light.

Rinse in PBS three times for 10 minutes each at room temperature, still protected from light. To mount the slides, add two drops of anti-fade medium with DAPI on each slide, cover with a coverslip, and store at four degrees Celsius until ready to image. Frontal bones of control embryos were positive for pSmad1/5/9, downstream BMP signaling factors, and Ki67, a cell proliferation marker.

In neural crest-specific, constitutively activated BMPR1A mutant embryos, however, pSmad1/5/9 levels were increased while Ki67 levels were decreased. Furthermore, more apoptotic cells were observed in the frontal bones of mutant embryos than those of control embryos. Coronal cryosections of gelatin-embedded heads clearly show GFP signal and RFP signal from a tdTomato cassette in the nasal bone and nasal tissues, demonstrating that gelatin does not interfere with fluorescent signals.

When these sections were immunostained for SOX9 or Osterix and E11/Podoplanin, good-quality sections were obtained from most of the three-week hard tissues. On the other hand, with three-month-old samples, good-quality sections were only obtained in some of the hard tissues, including the trabecular compartments of the femur, nasal tissues, and the skull, including the nasal-premaxilla suture and surrounding bones of the head. SOX9-positive cells were detected specifically in the chondrocytes of the growth plate and the joint from the femur and the nasal septum.

In the three-week-old incisor, SOX9 was detected in the mesenchymal cells. Osterix and E11 double staining results show that Osterix was detected in osteoblasts, while E11 was detected in osteocytes of bones from the femur and the head. In the three-week incisor, Osterix was positive in odontoblasts, while E11 was positive in follicle mesenchymal cells.

Please do not overfix samples with 4%PFA. For sectioning of undecalcified hard tissues in gelatin, best temperature is minus 25 degrees Celsius and lower. Following this procedure, levels of fluorescence can be quantified as described in our protocol.

Those measurements will provide informative data to show the difference of the relative level of protein between different groups. After its development, this technique paved the way for double or triple staining to get co-expression patterns of different proteins. 4%PFA is hazardous.

It may cause skin allergies, serious eye damage, organ damages, or cancer. Please use personal protective equipment, and wash skin thoroughly after handling.