Standardized Bone Dissection Protocol for WT and Transgenic Mice (UMKC Musculoskeletal Histology Core)

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1 Standardized Bone Dissection Protocol for WT and Transgenic Mice (UMKC Musculoskeletal Histology Core) For the standard analysis of the skeleton of transgenic mice, the bones that will be fixed are the hindlimbs, forelimbs, vertebrae and calvaria. The standard histomorphometric analysis is usually performed on the hindlimb and the lumbar vertebrae (L3 and L4 are normally used for analysis). The following protocol provides instructions on how to dissect and fix the tissues for our standard analysis procedures. The Importance of Careful Dissection and Fixation of Tissues Careful dissection following a standardized procedure is important to ensure consistency of the experiments and reduce variation. Good fixation is essential to prevent autolysis (i.e. tissue breakdown from enzymatic activity in the tissue) and putrefaction (breakdown of tissue by bacterial processes). Remember that the quality of everything you do later (histology, immunostaining, etc.) depends on how well you perform the dissection and fixation of the tissues. Some Key Points to Remember When Dissecting Tissues for Histology: Work quickly and only sacrifice and dissect one mouse at a time. Get the tissues into fixative as soon as possible after the animal is sacrificed to ensure good tissue preservation. Make sure you use an adequate volume of fixative for the tissues. Fixation may be incomplete if you do not use enough. A rule of thumb is to use a volume of fixative that is at least 20 times the displacement volume of the tissues (see section on fixation for more information on suggested volumes). Ideally, the thickness of the tissues to be fixed should not exceed 5mm (the rule in clinical labs is 3-4mm, but sometimes this is impossible with undecalcified bones). Once you have finished dissecting an animal, place the tube(s) with the fixative and tissues on an orbital shaker plate or gentle rotator, as this gentle agititation will greatly facilitate the penetration of the fixative into the tissues. Do not place tissues into plastic histology cassettes until AFTER fixation as this may reduce the penetration of the fixative. Good communication between the dissecting team and histology lab will help assure success. Important Definitions: Anterior (ventral) Posterior (dorsal) Medial Lateral Left Limb Right Limb = front of the body = back of the body = towards or at the midline of the body. = away from the midline of the body. =this refers to the mouse s left limb. (i.e. if the animal is lying belly down, it would be on the left and if it is lying belly up, it would be on the right). = this refers to the mouse s right limb. (i.e. if the animal is lying belly down, it would be on the right and if it is lying belly up, it would be on the left).

2 Dissection Procedure: 1. Humanely euthanize the animal according to the procedure in your approved animal protocol. 2. Decapitation is recommended in addition to your euthanasia method to ensure that the animal is dead before you continue with dissection. 3. Normally, the sequence for dissection is hindlimbs, followed by forelimbs, followed by spine, followed by calvaria. 4. For each mouse, prepare two 40ml tubes of fixative. The left hindlimb and forelimb will go into one tube, the right hindlimb, forelimb and the calvarium will go into another tube. The spines will be pinned to a styrofoam board to flatten them and floated in a large container of fixative overnight to fix (see section on spine dissection). Figure 1 shows a summary diagram illustrating the dissection technique Hindlimb Dissection 1. Wet the fur of the animal with 70% ethanol to prevent the fur from flying around during dissection. 2. With the animal lying on its side, remove the skin over the right thigh and lower part of the leg using forceps and sharp scissors. 3. Cut the muscle on either side of the hip joint as indicated in figure Carefully dislocate the femur out of the hip joint, supporting the bone while you do so. You should be able to see the ball joint. Cut the remaining ligament/muscle attachments to free the limb. Our standard procedure is to cut off the top of the femur just below the ball joint to allow fixative to penetrate into the bone (see figure 1). The foot should be cut off just above the ankle to allow fixative to penetrate into the tibia. NOTE if your transgenic mouse model has a very low bone density, it may be too risky to dislocate the femur as it may snap during this procedure. In that case, you should just cut the bone as close as possible to the hip joint. 5. Dissect away the bulk of the muscle on the femur and tibia using curved scissors, taking care not to disturb the knee joint (the curved scissors will help you to avoid damaging the periosteum). You should leave 1-2mm of muscle around the bone to help the sections adhere to the slide. 6. Immediately place into fixative, 7. Repeat procedure for left hindlimb. 8. Remember to place the left and right legs into different tubes so that you will know which one is which. At this stage you can also use india ink or mercurochrome to mark the tissues or to mark which is the medial surface, etc.

3 Figure 1

4 Forelimb Dissection 1. Remove the skin over the right shoulder and upper arm. 2. Cut the muscle around the scapula to separate it from the trunk of the animal. 3. Cut through the clavicle, which is the only point at which the forelimb attaches to the skeleton (see figure 1). 4. Cut any remaining muscle that attaches the forelimb to the trunk to release the intact forelimb, including the scapula. Make sure you avoid cutting near the shoulder joint, as this is the place where the analysis is usually performed. 5. Remove any remaining skin from the lower part of the forelimb and cut through the ulna and radius as close to the wrist as possible (see figure 1). 6. Place into fixative. 7. Repeat procedure for left forelimb. Spine Dissection 1. Remove any remaining skin from the carcass. 2. Take out the viscera and internal organs (NOTE some investigators may also want to fix internal organs such as the kidney, liver, etc, depending on the requirements of their experiments). 3. Remove the front of the rib cage and trim ribs to 2-4mm either side of spine (see figures 1 &2). Make sure you leave the white membrane-like ligaments on the ventral side of the spine, as these are important for orientation of the tissue during embedding. 4. The animal should already have been decapitated, the limbs removed and the ribs trimmed. Next, remove any remaining loose soft tissues. You should be left with the intact spine, pelvis and tail. 5. The spine must be flattened during fixation so that the lumbar vertebrae can be sectioned in the same plane (see fig 2). This is done by pinning the spine down to a styrofoam or cork board ventral side down (i.e. the side with the white ligament) and floating this upside down in a large container of fixative (e.g. large sandwich box). 6. Fix overnight in 4% PFA at 4 C with gentle stirring. 7. After fixation in PFA, change the solution to 70% ethanol overnight with stirring. 8. After this, the spine will need to be trimmed before processing and embedding (see fig 3). 9. Make another cut at the last thoracic vertebra (where the last rib joins) and at the top of the sacrum just below L6 (see figure 3) to remove the lumbar vertebrae. Make sure that you leave the top of the iliac crest (pelvis) as this is a very important landmark when identifying the correct vertebrae for analysis. 10. The lumbar section is the most important (fig 3 - boxed area between #3 and #4), as our standard histomorphometry analysis will involve the lumbar vertebrae (L3 and L4). However, you may also keep the cervical and sacral sections and tail if you want to. 11. Trim excess muscle from the lumbar spine segment. Make sure you leave the white ligament/membrane on the ventral side. Take care that you do not cut any bone when you are trimming (curved scissors are recommended).

5 (note see fig 4 for a diagram of the numbering of the lumbar vertebrae) Figure 2 Figure 3

6 Calvarial Dissection 1. Insert curved scissors at rear of skull (see figure 1) Make two circular cuts on the top of the skull (see figure 1), then lift out the calvarium 2. Place into fixative. Fixation 1. The fixative for the forelimbs, hindlimbs and calvaria is 10% neutral buffered formalin, which can be purchased ready made from several vendors (in reality the concentration of formalin in neutral buffered formalin is actually 3.7%). 2. Make sure you use formalin that has been stored properly (i.e. no evaporation), 3. The volume of fixative should be at least 20 times the tissue volume Examples: 1 mouse limb (0.5ml volume) requires at least 10ml fixative (even better to use 20ml) 1 intact 25g mouse requires 500ml fixative Several limbs, spine & organs requires ml 4. Fix the bone samples for 24h (calvaria) or 48h (limbs). Put your samples on an orbital shaker or rocker during fixation to help the fixative to penetrate more quickly. 5. The fixative for the spine is 4% PFA - see histology core PFA recipe for making up this solution. Some may also be available from the core After Fixation Before you proceed to decalcification of any samples consider whether you want to do any X-rays or micro-ct of the specimens, as this must be done before decalcifying. After the 48h fixation period, if the samples are not going to be decalcified or embedded immediately, you should store them in 70% ethanol for longer term storage (e.g. while you are collecting microct data, etc). After that, our standard procedure is as follows: Left Leg - this will be decalcified for 2-4 weeks in 14% EDTA for paraffin embedding (please refer to relevant protocol )

7 Right Leg this sample is undecalcified and will be plastic embedded in methyl methacrylate for analysis of fluorochrome labeling, mineral, etc. (please refer to relevant protocol). Lumbar Spine this sample is undecalcified and will be plastic embedded for Von Kossa/Goldner s staining, etc. (please refer to relevant protocol). Figure 4

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