Materials

In this and the following sections, most reagents or stains can be obtained from the representative vendors listed with telephone numbers and websites in Table 3.

1. Schiff's sulfurous acid leucofuchsin reagent: This can be prepared using items 3-6 and 8-10 as described in Subheading 3.1., or a premade solution can be purchased from one of the vendors listed in Table 3. (Sigma-Aldrich: cat. no. S5133; Fisher Scientific, cat. no. 5532-5001; Thomas Scientific, cat. no. 692-A-1; VWR Scientific Products, cat no. JTU973-1.)

2. 5 N HCl: Add 215 mL concentrated acid to 285 mL deionized water.

3. 1 N HCl: Add 40.5 mL concentrated acid to 459.5 mL deionized water.

4. 0.01 N HCl: Add 1 mL of 1 N HCl to 99 mL deionized water.

5. Basic Fuchsin (pararosaniline chloride, certified, C.I. 42500). Comes as a dry green powder or as small green crystals with a gold metallic luster (see Note 1).

6. 10% Sodium metabisulfite (Na2S2O3; Sigma-Aldrich, cat. no. S-1516) or 10% potassium metabisulfite (K2S2O3; Sigma-Aldrich, cat. no. P-2522): Dissolve 10 g in 100 mL deionized water.

7. Sulfite water (1% sodium metabisulfite; make fresh daily): Add 10 mL of 1 N HCl to 90 mL of deionized water in a graduated cylinder and decant into a 250 mL-capacity screw-top bottle. Add 10 mL of a stock 10% solution of sodium metabisulfite to another graduated cylinder containing 90 mL of deionized water. Add this solution to the acid solution in the bottle, which generates 200 mL of

Table 4

Alternative to Chromic/Sulfuric Acid for Cleaning Slides and Other Laboratory Glassware

Table 4

Alternative to Chromic/Sulfuric Acid for Cleaning Slides and Other Laboratory Glassware

Product

Catalog no.

Vendor

Versa-clean concentrate

G4-342

Fisher Scientific

RBS-35 Cleaner

PL 2795G

VWR Scientific Products

DeContam

KC85D

ESPI

Micro 9G

9G31

International Products Corp.

Nochromix

32869-3

Aldrich Chemical Co.

Note: Environmental health and safety regulations restrict the use of chromic/sulfuric acid solutions for routine cleaning of laboratory glassware and glass slides because of the heavy metal contamination and low pH of these solutions. Listed here are several commercially available cleaning agents. Request Material Safety Data Sheets (MSDSs) from the vendor when ordering these materials. When diluting concentrates, remember to wear goggles or a face mask while slowly mixing solutions in a hood and allow to cool to room temperature before capping the container of the diluted solution.

Note: Environmental health and safety regulations restrict the use of chromic/sulfuric acid solutions for routine cleaning of laboratory glassware and glass slides because of the heavy metal contamination and low pH of these solutions. Listed here are several commercially available cleaning agents. Request Material Safety Data Sheets (MSDSs) from the vendor when ordering these materials. When diluting concentrates, remember to wear goggles or a face mask while slowly mixing solutions in a hood and allow to cool to room temperature before capping the container of the diluted solution.

SO2 rinse water. (Do not inhale noxious fumes!) Distribute the 200 mL into three covered Coplin jars.

8. Decolorizing neutral activated charcoal (100 mesh; Sigma-Aldrich, cat. no. 24,287-6 or Fisher Scientific, neutral Norite A).

9. Filter paper, qualitative grade.

10. Bottles and beakers, 100 mL and 250 mL, to mix and store reagents. Rinse storage bottles with 0.01 N HCl before using. Drain to dry.

11. Frosted end slides (e.g., Fisher Scientific, cat. no. 12-544-2) and No. 1 thin cover glasses.

12. Plastic dishes that will hold 72 slides for cleaning.

13. Methanol/formalin/acetic acid (MFA), 85 : 15 : 1 (v/v).

14. D.P.X resin (Bio/Medical Specialties, [email protected]).

15. Immersion oil and refractive index liquids from nD 1.520 to 1.580.

16. Staining glassware such as Coplin jars and staining trays.

17. Liquid N2 or blocks of dry ice (and protective gloves!).

18. "Subbed" slides for en bloc Feulgen staining. Cell squashes and dispersal of en bloc-stained tissues are best done on frosted end slides that retain penciled labels and carry a thin film of chromed gelatin as a protein "glue" to ensure that tissues will adhere to the slide and not to the cover glass. Slides for subbing should be scrupulously clean before applying the gelatin film.

To clean the slides:

a. Use Micro-90 cleaning concentrate (or other product listed in Table 4) to prepare a 1% working solution by adding 20 mL of the concentrate to 2 L of deionized water.

b. Soak 72 slides for 2-3 h (or overnight) in a 1% Micro-90 cleaning solution. Wash the slides thoroughly in several changes of deionized water until the film of water flows smoothly over surface of the slide and does not break into beads or streaks (i.e., the surface "wets" perfectly).

To coat the slides with gelatin:

a. Add 2 g of chrome alum [chromium potassium sulfate, CrK(SO4)2T2H2O] to 40 mL of deionized water. Stir to dissolve completely. This 5% stock solution can be stored indefinitely.

b. Sprinkle 0.5 g of gelatin (e.g., Knox brand from supermarket) onto the surface of 100 mL of deionized water in a 250-mL Erlenmeyer flask at room temperature.

c. Incubate the mixture at 60°C to dissolve the gelatin (approx 30 min).

d. When the gelatin is in solution, use a 1-mL pipet to blow 1 mL of 5% chrome alum into the warm gelatin.

e. Decant the "chromed" gelatin solution into a Coplin jar and allow to cool to room temperature before use.

f. Dip each slide into the gelatin solution for 2-3 s.

g. After draining excess solution on the rim of the Coplin jar or onto a piece of absorbent paper, allow coated slides to dry vertically by leaning them against a surface that will keep them at about a 45° angle. Dry overnight. Store the subbed slides in a covered, dust-free slide box.

19. Parlodion (Fisher Scientific Co., cat. no. P35-100): Parlodion is a synonym for pyroxylin (a purified form of nitrocellulose) and is often called celloidin and may be obtained in the form of short, amber-colored strips. Caution! Because this is a DOT Class 4.1 flammable solid, due care must be taken to keep it in a flame safety cabinet or under a chemical hood. Keep in the dark; light causes deterioration. To make 0.25% Parlodion stock solution:

a. Add 0.25 g of dried pyroxylin strips to 100 mL of a 1 : 1 (v/v) mixture of ethyl ether and absolute ethanol in a glass bottle with a stopper.

b. Shake for 3-5 min to coat individual strips with the solvent.

c. Keep the bottled solution either in a flame cabinet or under a hood. It is flammable!

d. Parlodion strips take 3-5 d to dissolve completely.

3. Methods

3.1. Preparation of Schiff's Reagent

1. Add 10 mL of 1 N HCl to 100 mL of distilled water.

2. Add 1.0 g certified Basic Fuchsin (pararosaniline chloride) and shake for 10-15 min.

3. Add 10 mL of 10% sodium metabisulfite solution and shake vigorously for 2-3 min.

4. Let stand at room temperature until the solution turns straw colored, approx 2-3 h. Shake frequently during this period.

5. Place the bottle in a dark cabinet for 48-72 h (see Note 2).

6. Swirl the solution at least twice a day to suspend the contents.

7. After the desired time, add 0.5 g neutral activated charcoal per 100 mL of reagent.

8. Shake the suspension vigorously for 1-2 min.

9. Filter quickly through coarse filter paper (qualitative grade). The filtrate should be glass clear and colorless. If not, repeat steps 7-9.

10. Label 100 mL bottles, filled to the top and closed tightly to minimize air space.

11. Store in a refrigerator until needed (see Notes 3 and 4). Bring to room temperature before use (see Note 5).

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