Talk:Kubke Lab:Protocols/Gelatin Subbing

(Note: Have written protocol on front page --MF Kubke 18:12, 17 January 2011 (EST))

This is a work in progress, expect changes.

Protocol: Gelatin Slide Subbing

This is the procedure used to coat microscope slides with a gelatin film. This aids in the adhesion of tissue sections to the microscope glass and may prevent the sections falling of during staining. The pre-wash procedure can be skipped if slides purchased as 'pre-cleaned' (but not recommended) are coming out of a fresh new box and the slides are clean. Slides are kept in a staining rack for the entire protocol. Ensure that each slide is separated from the next within the rack.

Solutions

Acid alcohol solution

70% Ethanol
5% HCl
25% dH2O

Acetone

Use pure acetone.

Subbing solution

1. Heat 800ml dH2O to 40°C in a 1L beaker on a magnetic stirrer (excess heat will cause protein denaturation).
2. Keep the temperature in check temperature with a thermometer to ensure you are not going to destroy your gelatin before adding gelatin.
3. Slowly (giving it time to dissolve) add 4g of powdered Gelatin (.5%V

(Note: what do you mean?--MF Kubke 01:29, 17 January 2011 (EST))). Add the gelatin slowly while the stirrer is spinning on a high speed. This will prevent it from sticking to the sides of the beaker.

1. Once the gelatin is fully dissolved, add .4g of Chromium Potassium Sulfate(.05%V)<-

(Note: what does this mean?--MF Kubke 01:29, 17 January 2011 (EST)).

1. Reduce the stirring speed (high speed spinning puts bubbles in the solution)

(Note: Need to check the rest of the protocol with Felipe. This is not our standard protocol.--MF Kubke 01:29, 17 January 2011 (EST))

Pre-wash procedure

1. Pre-clean and warm up slides by leaving them in a bucket of warm/hot water with dish washing detergent (tap water is fine) and leave for 5 minutes agitating the mixture once a minute. Scrub the slides with a sponge, and place them into a slide holding rack.
2. Rinse well with hot tap water, followed by distilled water.
3. Remove excess water by tapping the racks against a paper towel.
4. Rinse in two big Petri dishes slide staining jars filled with dH2O, and remove excess water by tapping against a towel again.
5. Submerge rack in acid alcohol for one minute. (regular alcohol if no acid alcohol is available)
6. Remove excess solution by tapping on a paper towel.
7. Submerge rack in acetone for one minute.
8. Remove excess acetone by tapping on a paper towel.
9. Place slide rack (covered)and allow to evaporate, or manually dry the glass slides using Kimwipes or other lint-free tissue.

Subbing procedure

(Note: Need to check with Felipe. This is not my standard protocol)

Notes

• Keep all your equipment as dust free as possible.
• Drying times need to be quick so use a well ventilated, dry, warm and clean atmosphere to dry.
• The gelatin denatures at temperatures above 55°C so be careful never to heat anything that comes into contact with the gelatin more than this.

*Place a couple of paper towels between your heating platform and your beaker or flask to reduce the chance of the glass getting too hot.