Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

This protocol describes a canonical method to understand the critical genes controlling osteoclast activity in vivo. This method uses a transgenic mouse model and some canonical techniques to analyze skeletal phenotype.

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Genetically engineered mouse models are powerful tools for studying the mechanisms of human disease in vivo. Analyzing the skeletal phenotype of mice is the basis of skeletal research. This particle describes some typical techniques for analyzing the skeletal phenotype which may be interesting for those who are new to skeletal tissue research.

When attempting this particle keep in mind that animal experiments take time, so collect as many high quality samples as possible during each experiment, even if you don't need them in the short term. Begin by placing a euthanized mouse in a supine position and gently dislocating the bilateral hip joints by hand. Use ophthalmic scissors to vertically cut off the skin from the distal tibia and then remove all skin from the hind limb.

Cut off the articular ligament of the right hip joint and knee joint with scissors to separate the hind limb. Then cut the bone at both ends to fully immerse the bone in 4%PFA. Cut the trochanter and the junction of the fibula.

Immerse the hind limb in 4%PFA, keeping the right hind limb for paraffin sectioning. Cut the articular ligaments of the left hip and knee joints with scissors and gently remove the soft tissue. Separate the tibia and femur and immerse them separately in 75%ethanol.

Keep the femora for micro CT scanning and the tibiae for calcein and alizarin red double labeling. To prepare the paraffin sections, gently wash the fixed right hind limb three times with PBS for 10 minutes per wash. Then decalcify it in 15%EDTA with an ultrasonic decalcifier for three to four weeks until the bones can be bent, replacing the decalcifying fluid every other day.

After decalcification, wash the specimens three times with PBS and immerse them in 75%ethanol at four degrees Celsius overnight. On the second day, sequentially immerse specimens in 95%ethanol, 100%ethanol and xylene for one hour each. Immerse the specimens in half xylene and half paraffin for 30 minutes, then in paraffin at 65 degrees Celsius overnight.

To embed the specimen, submerge it in paraffin, placing the femur and tibia at a 90 degree angle. When the paraffin has fully cooled, remove the specimens from the embedding tank. Number and store them at negative 20 degrees Celsius overnight.

Bake the paraffin sections at 65 degrees Celsius for 30 minutes, then de wax them by immersing them in xylene for 10 minutes. Immerse the sections three times with fresh xylene each time. Rehydrate the sections by immersing them sequentially in 100%ethanol, 95%ethanol, 70%ethanol and distilled water for five minutes each.

Prepare the staining solution using the TRAP staining kit and warm it to 37 degrees Celsius. Add 50 to 100 microliters of staining solution to each section and incubate them in a 37 degrees Celsius humid chamber for 20 to 30 minutes. Check the staining status of the osteoclasts under a light microscope every five minutes until red multinucleated osteoclasts can be seen.

Then end the reaction with water. Counterstain the sections in hematoxylin solution for 30 seconds and create a stable blue color by immersing them in 1%ammonia solution for one minute. Then rinse them in slowly running tap water.

Mount the sections using cover slips with neutral balsam and dry them overnight. Capture three to five fields of view with a microscope and analyze the trabecular perimeter with image J.Use the straight line tool to measure the length of the scale bar as L1.Then use the segmented line tool to measure the length of the trabecular perimeter as L2.Calculate the physical length and count the number of TRAP positive cells with more than three nuclei. After fixation, gently wash the tibiae three times with PBS and sequentially immerse the specimens in 95%ethanol, 100%ethanol and xylene for five minutes each.

Immerse the specimens in acetone for 12 hours and half acetone and half resin for two hours, and in pure resin in a drying oven overnight. Add pure resin into a suitable silica gel embedding tank and place the specimens in the tank avoiding bubbles. Polymerize the resin in a drying oven at 60 degrees Celsius for 48 hours.

Cut the specimens into five micrometer thick sections continuously with a rotary microtome and store the rest of the samples with desiccant at room temperature. Adhere the sections with tweezers in a drop of 75%ethanol and mount them with cover slips using neutral balsam. Capture the red and green fluorescence labeling with a fluorescence microscope.

Osteoclasts-specific Stat3 deletion mice were generated to study the influence of Stat3 deletion on osteoclast differentiation. Femoral reconstruction and quantitative analysis by micro CT indicated that the bone mass of the Stat3 Ctsk mice was increased compared to wild type mice. Histo morphology of the femora from wild type and Stat3 Ctsk mice was examined via H and E staining.

Osteoclastogenic activity was detected using TRAP staining. Osteoclasts are large TRAP positive cells with multiple nuclei. The number of TRAP positive osteoclasts was lower in Stat3 Ctsk mice, compared with wild type mice, indicating that Stat3 deficiency impaired osteoclast formation.

Osteogenesis was measured with calcein and alizarin red double labeling. The area between the calcein and the alizarin red fluorescence represents newly formed bone. The deleted Stat3 in osteoclasts did not influence bone anabolism.

Sagittal paraffin sections in which the cartilage later were symmetrical and that showed that clear M-shaped line was used here. For the studies, we'll include more characteristics of skeletal system, such as mechanical properties.

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Journal (Biology)

Journal (Biology)

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

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