Preparation of Paraformaldehyde Solutions
(Contributed by Dr. Cecil Fox, Molecular Histology Labs)
Obtain reagent grade paraformaldehyde powder (e.g., Fluka). Weigh out 40 grams of the powder in a chemical fume hood and pour the powder into 2000ml Erlenmeyer flask. Make to one liter with distilled/deionized water. Add a stirring bar. Heat the liquid to 70ºC with stirring IN A CHEMICAL FUME HOOD. Add 1 drop of 10M KOH or 10M NaOH. The solution should become clear immediately after addition of the base. Allow the solution to cool to room temperature. Adjust the pH to 7.3. If the solution is not to be used immediately, add 50 ml of pH 7.3, 0.2M McIlvanies citrate buffer per liter. Use the fixative at room temperature by adding the tissue to 20 volumes of fixative. Smaller tissue pieces (3mm thick) fix better.
Artificial Tissues From Cells
(Contributed by Dr. Cecil Fox, Molecular Histology Labs)
Purpose
To produce a cell suspension that allows the separate study of single populations in an inert medium. A plasma clot containing the cells is prepared and fixed in formaldehyde or other agents. The clot is embedded in paraffin and sectioned on a microtome and sections are mounted on slides. The cells may be from cultures, scrapings, body fluids, or from washes of organs.
Method
- Suspend 1x106 cells in 1ml of medium or PBS in 12 ml conical centrifuge tube. Gently spin the tube at 1000 rpm for 30 seconds. Remove as much of the supernatant fluid as possible.
- Add 1 ml of human plasma. Resuspend the cells by low speed vortexing.
- Quickly squirt 250 ml of topical bovine thrombin directly into the plasma. We prefer USP topical thrombin sold as Thrombogen™ #1911 by J&J Medical, Arlington, TX 76004. We dilute this to contain 2000 units/ml.
- Allow the resulting clot to fully form for about 10 minutes.
- Add 10 ml of fixative at room temperature. Invert the tube to be sure the fixative surrounds the clot.
- We suggest for fixation Parafix™ from Molecular Histology Laboratory for use in situ hybridization or MHL Munofix for immunohistochemistry.
Reference: Cottler-Fox, M., and Fox, C.H. Examining cells for infectious agents: a novel approach. J. Inf. Dis. 164:1239-1240. 1991
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