General NMF Histology Protocols
Full Workup
Histology for all mutants should begin with a “full workup”, either alone or in conjunction with other procedures. The full workup consists of:
1) Observe the mouse’s phenotype and record
2) Perfusion fixation with Bouin’s fixative- intracardial infusion after anesthesia.
3) Immerse mouse in Bouin’s for about 7 days.
4) Harvest tissues into 9 cassettes to be sent to histology dept.:
1) Brain (6 coronal sections; label with ‘B’)
2) Salivary gland, trachea/ thyroid, thymus, skin
3) Pituitary/ears in situ
4) Leg
5) Spleen, heart, liver, lung, kidney, adrenal gland
6) Stomach, duodenum, jejunum, ileum, cecum, colon
7) Bladder, pancreas, male organs (teste, epididymis, prostate seminal vesicle); Female organs (uterine body/horns, ovaries)
8) Spine/spinal cord – (5 sections; label with ‘S’)
9) Eyes removed before perfusion and fixed ~24hours in Bouins
5) H&E should be done on all tissues, and luxol-fast blue/ cresyl violet (LFB/CV) on brain and spinal cord.
Motor Deviants
Myosin Antibody (AB) Labeling– Performed on frozen sections, muscle can be removed before perfusion.
NMJ Staining - For the neuromuscular junction stain, the muscles must be removed before perfusion fixation while the mouse is anesthetized, but can still be done with the full workup.
Lumbar Nerve Count- serial sections of paraffin-embedded spine (Bouin’s perfusion fix/ immerse 1 wk) from L3 to L6. MUST be consistent to get accurate nerve count.
Dissection Protocol: Remove all organs in abdomino-thoracic cavity. Peel aorta/ inferior vena cava away from spine exposing vertebral bodies. The area you want is even with adrenal glands. Starting at rostral end, remove vertebral bodies one by one exposing the spinal cord. After the cauda equina is exposed, you can see a ‘bulging’ area of the cord. This is the area to be serial sectioned. Remove this ½ inch area and mark caudal end with pencil.
Sectioning Protocol: Serial section whole tissue rostral to caudal, placing 5 sections per slide (5microns thick). When counting neurons, every 4th slide is counted allowing 4 different ‘reading frames’, in case 1 reading frame is missing too many sections. If sections are messed up, instruct to leave a space on slide.
Staining Protocol: Same as for myosin AB labeling, but use SMI 34 1:15,000 diluted as primary AB.
Seizure Deviants
Perfusion fix with Bouin’s (same as full workup). Paraffin-embed and obtain coronal serial sections through hippocampus, including CA1, 2, 3, and dentate gyrus.
Vision Deviants
Eyes may be processed in plastic, if necessary. The eyes of the mouse should be removed prior to perfusion, fixed in Smith/Rudt fixative, and brought to the histology lab within 24 hours. Serial sections on Bouin’s fixed eyes may also be performed. The ideal section for the eye is right through the center of the eye and optic nerve.
Hearing Deviants
Whole Ear Exam – allows examination of the inner ear for presence of otoconia and gross deformations in inner ear structure. View otoconia with polarizing lens.
1) Remove inner ears and fix 4-6 hrs in 10% neutral buffered formalin 2) Dehydrate – 2x70%, 95%, 2x100% EtOH, 1 hour each. Last step overnight.
3) Clear in methyl salicylate under vaccuum to remove air bubbles.
4) Store in methyl salicylate.
Cochlear Sections- Prepare same as full workup. Obtain serial sections transversing the cochlea in order to view organ of Corti, cochlear duct, etc.; H&E stain.
MOUSE PERFUSION WITH BOUIN'S FIXATIVE
Note: This procedure must be performed under a hood:
1. Set up the butterfly apparatus: Attach a two-way connecting valve with two 20ml syringes (one containing saline* and the other containing Bouin’s** fixative).
Note: make sure the valve for the Bouin’s is shut off to ensure that the mouse is initially perfused with saline.
2. Have small scissors and forceps readily available.
3. Anesthetize mouse with Avertin*** (.3 ml/10 gm/body weight). Use a 26 gauge needle.
4. Remove eyes using scissors, do not pluck, and place in a cassette in Bouin’s.
5. Pin mouse to angled board (head angled down) which is inside a container large enough to collect the drain-off of saline and Bouin’s.
6. Cut skin at thoracic area just below the rib cage.
7. Remove sternum to expose heart.
8. Snip the right atrium with scissors.
9. Insert butterfly needle into left ventricle (snip approximately 2mm of plastic off tip of butterfly to expose the needle and act as a gauge for entering the heart).
10. Perfuse with saline at a slow, steady pace.
11. Switch shut off valve to the saline syringe and perfuse with Bouin’s.
12. Open abdominal cavity to expose the abdominal viscera.
13. Peel skin back to expose the muscle tissue.
14. Remove skin and ears from skull, exposing the ear canals.
15. Open the top of the skull to expose the brain.
16. Submerge mouse in Bouin’s for approximately one week prior to trimming into cassettes.
* Saline: 4.25mg. NaCl/500ml de-ionized water.
** Bouin’s fixative: Obtain prepared fixative from the histology lab.
***Averdin: Obtained from Animal Health (tribromoethanol, Sigma Aldrich, USA).
