Protocol 10 Immersion method

Method

1. If tissues are derived from animals that were not perfused or from human autopsy specimens, put excised tissues/organs into fixative for 3-5 h (Bouin's) or overnight (10% neutral-buffered formalin) in intact form, then cut in half to allow for better infiltration of fixative if a large organ (e.g. liver, heart, muscle, spleen). Cutting is generally not required for mouse lymph nodes or thymus because the tissues are small. [For brain tissue, fix intact overnight (even if using Bouin's), then cut the next day.]

2. Let the tissues fix for an additional 3-5 days, depending on the size of the tissues (e.g. mouse/rat for 3 days vs. human autopsy for 5 days). Typically, the fixation is done in plastic beakers/jars.

If tissues are derived from perfused animals (see below), the postfixation time can be reduced to 1-2 days and the formalin is only 4% instead of the usual 10%. Post-fixations in Bouin's solution are at usual full strength.

3. Place tissues into cassettes for paraffin embedding. Label with a 'superfrost' marking pen which is resistant to alcohols and xylenes. It may be necessary to cut the tissues smaller with a scalpel to get them to fit. However, do not let the tissue pieces dry out at any point. Also, orient the tissues in the direction you will want them cut, then instruct the histotechnician to organize the tissues in the paraffin molds in the same fashion as they appear in the cassettes. (The bottom of the cassette is the cutting surface.)

4. Submerge the cassettes for a few hours in 50% EtOH/PBS and then proceed to dehydrations using an automatic tissue processor/ Alternatively, if fixed tissues will not go directly to embedding, dump out the fixative and replace with PBS. Change the PBS solution twice over the next day or so and store the fixed tissues in PBS at 4°C. These fixed tissues should be good for at least a few weeks, but add azide if keeping longer or if shipping unembedded tissues at room temperature.

sOur tissue processor is a Shandon, Inc. (model Citadel). This must be optimized for your own processor, but the conditions we use are: (a) 50% ethanol/PBS for 1 h; (b) 70% ethanol/water for 1 h; (c) 95% ethanol for 1 h; (d) 95% ethanol for 1 h again; (e) 100% ethanol for 1 h; (f) 100% ethanol for 1 h again; (g) xylene or xylene substitute (Hemo-De from Fisher, cat no. 15-182-507A or Histosol from National Diagnostics, cat. no. HS-100) for 1 h; (h) repeat xylene or xylene substitute for 1 h; (i) paraffin at 57-61 °C for 1.5 h; (j) repeat paraffin at 57-61 °C for 1.5 h. Note that isopropanol can be substituted for ethanol in all of these steps.

Protocol 11. Perfusion method

For paraffin immunocytochemistry, the optimal solution composition should be either: 4% formaldehyde, buffered with PBS to pH 7.4-7.6 (4% formaldehyde/PBS) or 2% paraformaldehyde in PBS. The approximate volume required for perfusion by cardiac puncture is 100 ml per mouse and 200-300 ml per rat. An i.v. infusion system with a volving system and manometer is required, equipped with appropriate tubing and a 16-18 gauge needle attached for cardiac punctures.

Method

1. Animals are first placed into deep anaesthesia, usually using Nembutal.

2. Lift the skin over the mid-epigastric region and make an incision with scissors. Spread open the hole with the scissor blades and cut horizontally to reveal the diaphragmatic muscle. Carefully cut the diaphragm away from its attachments. Cut the ribs and lift the sternum, revealing the heart. Insert the needle, hooked to the infusion bottle containing PBS without fixative into the left ventricle. Immediately cut a small hole in the right atrium or ventricle with scissors to relieve pressure. Start the flow of PBS into the left ventricle with pressure set at 100-120 mmHg. (The animal should be in a pan to catch the fluid that will drain from the right side of the heart.) Continue infusion of PBS at 100-120 mmHg until essentially all blood has been removed from the vasculature: i.e. the flow from the right ventricle is clear PBS and not blood.

3. Switch to the fixative infusion bottle and infuse fixative until the body of the animal becomes stiff (about 10 min for mice and 15-20 min for rats).

4. Discontinue the infusion, and excise the tissues of interest.

5. Place the tissue into post-fixative which is either the same formalin solution used for perfusion or Bouin's solution.

6. If sufficient material is available, it is often advisable to split it into two portions. Post-fix one of the pieces in Bouin's solution, which is generally the better fixative for Bcl-2 protein family immunocyto-chemlstry (Sigma Dgn.; cat. no. HT10-1-128). Post-fix the second piece in 4% buffered formalin for immunostaining with other antibodies or for DNA fragmentation analysis by the TdT end-labelling method (34). (Bouin's solution fixation results in non-specific DNA breaks.)

"Tissue processing and paraffin embedding is accomplished as in Protocol 10 above.

6.1.2 Paraffin embedding

Do not overheat or overdehydrate the samples. Keep the temperature of the paraffin no higher than 65 °C. We use the 56-60 °C melting-temperature paraffin from Surgipath, Inc. (cat. no. El-600) or equivalent. (Note: The two pieces fixed differently can be embedded in the same paraffin block under the same patient/experiment number. For example, we have adopted the convention of placing the Bouin's-fixed piece at the top and the formalin-fixed piece at the bottom of the slide.)

6.1.3 Sectioning

Paraffin sections 3-5 p,m are prepared and mounted on poly-L-lysine coated slides or Fisher-Superfrost-plus slides (generally 50-200 slides can be obtained per block, but of course it depends on the size and thickness of the tissue specimen).

If the paraffin samples are small, 2 or 3 separate patient samples or experimental animal samples can be placed adjacent to each other on the same glass slide so as to minimize the labour of immunostaining.

6.1.4 Solutions

Modified PBS. NaCl 7 g/litre

NaH2P04 (X H20) 1.38 g/litre K2HP04/anhydrous 5.44 g/litre adjust pH to 7.4-7.6 4% Neutral-buffered formalin/PBS. add 108 ml of 37% formaldehyde bring volume to 1 litre using modified PBS readjust pH to 7.4-7.6 with 10 N NaOH 10% Neutral-buffered formalin/PBS

first make 2 X PBS solution by combining the salts described above into 0.5 litres instead of 1 litre. Then mix:

500 ml of 2 X PBS

270 ml of 37 % formaldehyde

230 ml of dH20

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