Adjust pH to 7476 with 10 N NaOH 62 Prestaining sample preparation

Protocol 12. Deparafinization procedure Method

1. Pre-warm the slides in an oven at 55°C for 1-3 days. This enforces attachment of the tissue to coated slides. (If you receive your slides from an outside source, check whether this has already been done.) The slides should be placed into a Wheaton glass slide rack for this procedure.

Any oven can be used for this. A standard vacuum oven found in most molecular biology laboratories is suitable. We typically hook to house vacuum line and turn on the vacuum, which helps to keep the temperature constant (~15 mmHg).

2. Remove the slides from the oven. Then pre-warm the oven to 65°C before reinserting.

3. Melt the paraffin at 65°C for 20 min (the drops of paraffin should be seen at the bottom of the slides).

4. Set up three separate glass containers (Wheaton/Fisher, cat. no. 08812) containing xylene or xylene substitute (such as Hemo-D from Fisher Scientific, cat. no, 15-182-507A) and transfer the slides while still in the glass slide rack sequentially to each jar for 2 min each. Agitate the slides up and down a few times during each transfer before submerging for 2 min, then agitate up and down before transferring to the next container of xylene. Shake off drops before transferring to the next solution.

5. Immerse slides in 100% ethanol for 15 sec.

6. Place into 3% H202 in methanol for 30-45 min.

7. Next, transfer to 95% ethanol for 20 sec.

8. Then transfer to 70% ethanol for 20 sec.

From this point on, keep the slides moist. DO NOT let them dry out.

Protocol 13. Antigen retrieval (acid treatment)

Microwaving in acidic solutions is used to enhance antigen reactivity.3


1. Transfer slides to four polyethylene Coplin 'jars' (Fisher Scientific, Inc.; cat. no. 08-815-10) and place these jars at the four corners of a plastic box. Each container should have 10 glass slides. Insert 'blank' glass slides if your experiment involves fewer than 40 slides.

2. Add acidic solution (pH 5.5-6.0) into the containers until the slides are just covered. Also put tap water into the plastic box until the bottom is covered.

3. If your microwave does not have a rotating stand, after each pulse of microwaves change the positions of the flasks (it is convenient to simply move the container clockwise or counter-clockwise so that each of the coplin jars will have occupied all four corners of a square by the end of the procedure). Also gently agitate each container after each heating to ensure uniform heating of the fluid

John C. Reed et al. Protocol 13. Continued

4. For our oven (General Electric; model JE1453H-001), we heat as follows:

Power setting Time (min) Desired temperature (°C)

In general, however, the goal is to bring the solution in which the slides are submerged close to a boil (~90-95°C) without boiling. (If boiling does occur for a few seconds, however, it is usually not deleterious.) Once brought to a temperature of 90-95°C, the sample need not be held at this temperature. Simply let cool it to room temperature.

5. Let the containers cool at room temperature for 15 min.

" Microwaving should be performed at a slightly acidic pH. The dH20 at our institution has a pH of —6.0 and we therefore use it directly. Alternatives include 10 mM citrate buffer at pH 6.0 or 50 mM acetate buffer at pH 5.5. These conditions should be optimized for your particular microwave and water source.

Protocol 14. Alternative antigen retrieval protocol

An alternative to microwaving is to treat the slides with more acidic solutions without heating. Though results are superior with microwaving, some tissue sections can detach from the glass microscope slides during microwaving (depending on how they were prepared) and therefore this alternative procedure is also provided here.


1. Incubate slides in 0.02 M citrate buffer (pH 3.2) for 2 h.

9: Methods of measuring Bcl-2 family proteins 6.3 Immunostaining

Protocol 15. Immunostaining procedure


• primary antibodies. Polyclonal rabbit antisera to Bcl-2 family proteins or mono-clonals derived from mouse or hamster

• glass dishes with removable horizontal tray that holds slides (20 slides capacity; Fisher/Wheaton, cat. no 08-812)

• closed humidity chamber; prepared from plastic boxes with plastic pipettes (10 ml) fixed at the bottom with superglue. Put tap water in the bottom of the box

• flat-tipped forceps

• reagents for avidin-biotin complex preparation (Vector Lab. ABC-Kit Standard; cat. no:PK-6100)

• secondary antibodies. Biotinylated goat anti-rabbit IgG (Vector Labs, cat no: BA-1000) or biotinylated horse anti-mouse IgG (Vector Labs, cat no: BA-2000) or biotinylated sheep anti-hamster IgG (Vector Labs, cat no: BA-9100)


This procedure is optimized for final detection of antibodies using an avidin-biotin complex reagent and diaminobenzidine colorimetric reaction involving horseradish peroxidase. Other detection methods may require some modifications. Perform all of the following steps in a humidity chamber at room temperature.

Do not let the slides dry at any point. Keep covered with sufficient volume of various reagents (—500 jd per slide).

1. Pre-block by incubating in slides in either 0.1 M Tris (pH 7.6-7.8) or TSK [Tris/sodium chloride/potassium chloride (pH 7.6-7.8)] with 2% BSA, 1% normal goat, and 0.05% Triton X-100 for 1 h.a-ft

2. (a) Decant the pre-blocking solution and incubate with 0.4-0.6 ml of primary polyclonal antibody diluted typically 1:800 to 1:2000 in either 0.1 M Tris or TSK containing BSA and Triton X-100. Alternatively, 3-10 |xg/ml of anti-Bcl-2 murine monoclonal antibody or 2 n-g/ml of anti-Bcl-2 hamster monoclonal antibody in either 0.1 M Tris-BT or TSK-BT may be used. Incubate overnight at room temperature.0

(b) A negative control slide(s) should also be prepared using pre-immune serum of the same rabbit or, alternatively, normal rabbit serum. Use isotype- and subclass-matched control monoclonals when using anti-Bcl-2 monoclonals (Dako, Inc., Santa Barbara, CA). If no negative control antibodies are available, perform immunostaining using only the secondary antibody, without a primary antibody.

(c) An excellent control entails pre-adsorbing the antibody with excess antigen (peptide or recombinant Bcl-2 protein). For peptides, we typically add 10 (xl of antiserum to 1 ml of TSK-BT and 10 p.l of a solution containing the appropriate Bcl-2 peptide at 1 mg/1 ml. The antigen and antibody are incubated at 37°C for 2 h before use.

3. The next day, wash slides three times in PBS for 5 min each using plastic Coplin jars. Agitate gently to wash. Do not put slides with different antibodies (or even different dilutions of antibodies) together in the same washing container.

4. Incubate with —0.5 ml of secondary antibody for 1 h in a humidity chamber.

(a) For polyclonal antisera: 3-5 (j.g/ml of biotinylated antibody from goat anti-rabbit immunoglobulins (Vector Lab.; cat. no. BA-1000) in 0.1 M Tris-BT or TSK-BT, with 0.5% normal serum.d (Usually 5 fxg/ml is used.)

(b) For mouse monoclonals: 2.7 jjig/ml of biotinylated horse anti-mouse IgG (Vector Labs, Inc.; cat. no. BA-2000) in either 0.1 M Tris-BT or TSK-BT with 0.5% normal serum.d

(c) For hamster monoclonals: 2.5-2.7 jig/ml of biotinylated sheep anti-hamster IgG (Vector Labs, Inc.; cat. no. BA-9100) in either 0.1 M Tris-BT or TSK-BT with 0.5% normal human serum.

5. Wash three times for 5 min each in PBS. (Keep slides separated into different Coplin jars if they received different secondary antibodies and/or different dilutions).

6. Incubate slides in a humidity chamber for 45 min in ~0.5 ml each of avidin-biotin complex reagent containing horseradish peroxidase (HRPase) (Vector Labs.; Vectastain Kit, cat. no. PK-6100). [This solution must be prepared in 0.1 M Tris (pH 7.4-7.8) 1 h before use!]

7. Wash with PBS three times for 5 min each. (All slides can be washed together at this point, regardless of primary or secondary antibodies or the dilutions of antibodies, with the exception that we generally take the additional precaution of washing separately any negative control slides, e.g. those that received pre-immune serum or peptide-adsorbed antibodies, etc.)

8. Develop the reaction product for 10-20 minutes in a humidity chamber by covering slides in DAB solution containing H202, as described below. (This solution must be prepared freshly each time!)

9. Wash slides in dH20 using plastic Coplin jars three times for 5 min each.

a We generally use 0.1 Tris (pH 7.6-7.8) but if high background is a problem, try TSK. The high salt concentration eliminates much non-specific immunostaining.

6 The use of goat serum assumes that the secondary antibody employed is derived from goat. If a different species is employed for secondary antibody production (horse, sheep, donkey, rabbit), then 1% of normal serum from that species should be used instead of normal goat serum. "If non-specific immunostaining is seen with polyclonal antisera raised against synthetic peptides, pre-adsorption of the antibody with the same protein used as a carrier (ovalbumin, KLH) can be helpful. The optimal amount of murine monoclonal depends on the quality of fixation, etc., but usually -10 mi/ml for Mab124 and ~2 n,g/ml for 407.

dInclude 0.05-0.1% normal serum from the same species that the tissue sections are derived from, e.g. if immunostaining human tissue then add human serum.

9: Methods of measuring Bcl-2 family proteins 6.4 Counterstaining and mounting

Protocol 16.

Perform all steps using glass slide racks. Counterstaining with methyl green may be preferred to haematoxylin when studying lymphocytes because of the scant cytoplasm (high nuclei/cytosolic ratio). However, methyl green will fade with time, especially if not kept in the dark. Generally, it is wise to photograph such slides within 2 weeks.


1. Rinse slides in tap water for 1 min.

2. Submerge slides in haematoxylin counterstaining solution (Mayer's Hematoxylin; Sigma, Inc., cat. no. MH5128) for 20 sec to as much as 5-15 min. (The optimal time depends on the source of haematoxylin and how fresh the solution is; it will take longer after many uses.) Alternatively, methly green can be used for counterstaining.3

3. Rinse slides In tap water for 1 min.

4. Place slides, still In the slide racks, into ~200 ml of tap water containing 5-7 ml of NH4OH (28-30%; Aldrich Chemicals, Inc., cat. no. 1336-21-6) for 15-20 sec.

5. Rinse in tap water again.

6. Rinse in dH20 for 1 min.

7. Dehydrate samples by submerging for 15-30 sec each in sequence into 70% EtOH, then 95% EtOH, then 100% EtOH, followed by three treatments with xylene or a xylene substitute.

8. Place a drop of acrytol mounting media (Surgipath; cat. no. MM-160) on to the slide over the tissue or cells, and apply a coverslip.

9. Let dry at room temperature for a few hours, while lying flat with the tissue.side up.

"(a) Counterstain in methyl green in a glass Coplin jar for 10 min at room temperature. (If the methyl green solution has be reused several times, increase the counterstaining time to 15-25 min.) (b) Wash three times in three changes of distilled water in a Coplin jar, dipping the slide 3-5 times each in the first and second washes, followed by 15 sec without agitation for the third wash, (c) Wash in three changes of 100% butanol in a Coplin jar, dipping the slide 10 times each in the first and second washes, followed by 10 sec with out agitation/dipping for the last wash, (d) Dehydrate in three changes of xylene for 2 min for each of the first two washes and for 5 min on the last wash, (e) Mount.

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