Kubke Lab:Protocols/Gelatin Subbing

(Note: version 12:16, 18 January 2011 is a signed version of the protocol --MF Kubke 18:16, 17 January 2011 (EST))

Protocol: Gelatin Slide Subbing

This is the procedure used to coat microscope slides with gelatin. This aids in the adhesion of tissue sections to the microscope glass to allow the sections to withstand the staining procedure.

Slides need to be prewashed prior to subbing with gelatin.

Two methods are provided (A and B) for cold and hot subbing. The two have been used successfully, but 'cold' subbing may be better for larger sections.

Solutions

Acid alcohol solution

For 100 ml solution

	Volume (in ml)
Ethanol	70
HCl 37%	5
ddH2O	25

Acetone

Use pure acetone.

Subbing solution

0.5% gelatin, 0.05% ChromAlum

For 1 L subbing solution

Chemical	Ammount
ddH2O	1 L
gelatin	5g
ChromAlum	0.5g

Procedure

1. Heat 800ml ddH2O to 40°C in a 1L erlenmeyer (see note below) - Make sure to control the temperature with a thermometer and adjust the heating plate so that the temperature of the water is stable.
2. Once the temperature is stable, slowly add the gelatin and let it dissolve, using a magnetic stirrer. The time it will take to dissolve will vary with the temperature.
3. Once the gelatin is fully dissolved let the solution come down to room temperature
4. When the solution is at room temperature add 0.5g of Chromium Potassium Sulfate (ChromAlum), stir gently until dissolved

(Note: Depending on the gelatin, the optimal temperature can be up to around 65C - Be careful to not heat above the optimal temperature so as to not denature the gelatin)

Glass Slide Pre-wash

1. Place the glass slides into hot (about 80 C) water with dish washing detergent
2. Leave for about 30 minutes, agitating frequently, or alternatively scrub the slides with a sponge
3. Rinse well with hot water, and place slides into a slide rack
4. Rinse well with ddH2O
5. Tap excess water onto a paper towel
6. Bathe the slides in the rack in Acid Alcohol Solution (alternatively use pure alcohol)
7. Tap excess liquid onto a paper towel
8. Bathe the slides in the rack in Pure Acetone (alternatively use pure alcohol)
9. Tap excess liquid onto a paper towel
10. Let slides dry in a clean dust-free area. You can warm the slides to about 40C for drying.

Subbing procedure A (Cold)

(Note: This procedure is the one that Felipe Medina uses, which he learned in his undergad lab in Chile. He says that it is better at preventing large brain sections from falling off the slide)

1. After the ChromAlum is dissolved, put the gelatin subbing solution on ice to bring down the temperature
2. Bathe the cleaned and dry slides in the Subbing Solution. Slides should be submerged for a minimum of 1 minute. {{Note|Make sure that there are no bubbles and that all slides are equally exposed to the gelatin solution
3. Remove the slides from the subbing solution and tap excess liquid onto a paper towel
4. Dry the slides in a dry, dust-free environment (you can warm them up as long as they are not heated above 40C)

Subbing procedure B (Hot)

(Note: This is the method I have used all my life --MF Kubke 18:07, 17 January 2011 (EST))

1. Warm up the gelatin solution to somewhere between room temperature and 40 C
2. Bathe the cleaned and dry slides in the warm Subbing Solution. Slides should be submerged for a minimum of 1 minute. {{Note|Make sure that there are no bubbles and that all slides are equally exposed to the gelatin solution
3. Dip the slides a few times before removing them from the subbing solution
4. Tap excess liquid onto a paper towel
5. Dry the slides in a dry, dust-free environment (you can warm them up as long as they are not heated above 40C)
6. You may wish to repeat this procedure and re-sub the slides after they are dried

Chemicals list and suppliers

Chemical	Description	Supplier	Catalogue Number
Gelatin