Staining of Tissue Slides

General Staining Guidelines

1. Slides are initially labelled with a lead pencil or solvent resistant marker but a sticky white label is attached before passing in. The label should fit the slide (even if they need to be cut) and not overlap the coverslip. Be careful to carefully check the information to eliminate the possibility of clerical errors!!   Information required: accession #, patient last name/first initial, your initials, and staining method (where appropriate)
2. For H&E’s you do not need to label the slide with “H&E” as this is the routine stain done on each slide.  Other staining methods should be indicated on the label.
3. For each stain you will do a slide and a control, all passed in to the instructor in a slide folder at the end of the lab. Please include a “lab report” for each of the methods (in pen). It takes approximately 24 hours for the cover slipping medium to set, so be careful when handling slides after mounting (see evaluation of slides).
4. Control slides need to be assessed and recorded in the QC binder each time they are run, with the date, the staining method, your initials, and whether or not they are acceptable
5. All controls should be labeled as “QC” with the name of the stain, the date,  and your initials.
6. Filter all stains before use.
7. Slides should never be allowed to dry during the staining process (held out of fluids for any length of time).
8. Always make sure the solutions completely cover the tissue on the slides while in coplin jars.  Dye solutions should cover the slots that separate slides, but not so full that labelling information is submerged.  Dehydrating alcohols and Formula 83 jars should be filled so that the whole slide is submerged when moving them through.
9. Take care not to damage sections while moving slides in and out of solutions with forceps.
10. Ensure that slides remain separate from one another while in coplin jars or their surfaces may stick to one another, resulting in loss of tissue.  Slides may be placed in criss-cross fashion but not back-to-back as this results in excess carry-over of reagent.
11. If there are no notes regarding the shelf life of a solution, the expiry date is one year.
12. Students may share stations but each person must make up their own working solutions. Each student must move their own slides through the solutions.
13. When moving through dehydrating alcohols and clearing agents to prepare for mounting, forceps should be submerged in each solution along with slides.  This will help to prevent water carry-over and contamination of finished slides.

Pre-Staining: Deparaffinize and Hydrate to Water

***NOTES:

• The alcohols used for hydration cannot be recycled. They must be discarded as they are contaminated with Formula 83 (or xylene)
• Formula 83/xylene must be used in fume hood
• Slides are now ready for staining

Post-staining: Dehydration and preparation for mounting

***Notes

• The order of the alcohols and formula 83’s is important when performing this procedure. The first alcohol will have the most water contamination. Since the goal is dehydration (no water remaining) it is important that the last 100% alcohol remain uncontaminated with water. Contamination with water will be visible as cloudiness and will cause issues with reading and interpreting slides. It can only be removed by “backing” up the slides from formula 83 into several uncontaminated coplin jars of 100% alcohol, and then into fresh uncontaminated formula 83 for mounting. Once 100% alcohols or formula 83 are water contaminated they must be replaced.
• Formula 83 (all) and dehydrating alcohols (used for dehydration only) can be recycled and should be poured into waste carboys for recycling
• Formula 83 must be used in fume hood