Talk:Kubke Lab:Protocols/Gelatin Subbing

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.

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). 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.
 * 4) Once the gelatin is fully dissolved, add .4g of Chromium Potassium Sulfate(.05%V)<-.
 * 5) Reduce the stirring speed (high speed spinning puts bubbles in the solution)

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.