Prisma & Film Staining Workshop. Application Specialist Mea Pelkonen

Size: px

Start display at page:

Download "Prisma & Film Staining Workshop. Application Specialist Mea Pelkonen"

Oliver Montgomery
5 years ago
Views:

1 Prisma & Film Staining Workshop Application Specialist Mea Pelkonen

2 Tissue-Tek Prisma

3 Tissue-Tek Prisma Always program the Prisma in the following order: 1. Edit solution names Check if desired solution names already exists Up to 100 solution names can be stored 2. Edit solution configuration Up to 50 configurations can be stored 3. Edit program Up to 50 programs can be stored

4 Tissue-Tek Prisma Edit Menu

5 Tissue-Tek Prisma Edit solution names Add solution names if not there already Change existing solution names 5

6 Tissue-Tek Prisma Edit solution configurations Start with configuration set-up Make the configurations by filling in the solutions in the template Choose management method Adjust station colors and parameters

7 Tissue-Tek Prisma Edit solution programs Name program Choose the configuration you want to use Start programming 7

8 Do s and Don ts Do: Program as much start stations as possible Program as much washing stations as possible Program the steps in a configuration on a physically short distance of reagent containers Try to avoid the use of short timing steps in a program Avoid, if possible, the use of one reagent vessel for two or more protocols Don t: Open the cover without software intervention Use the exact option in the program if not necessary

9 Reagent exchange Rotation/Replacement of Solutions All staining solutions or reagents have a definite lifetime Use the appearance of the sections and determine rotation/replacement of solution schedules depending on work volume, type of solutions used, and mode of staining (automatic or manual). Wash and rinse each staining vessel at the time of rotation Inadequate washing or rinsing can leave residues that can alter the chemical nature of new solutions placed in the vessel. Alcohol dehydration/clearant exchange A general rule is to rotate the final anhydrous alcohol when it acquires a pink cast from eosin carryover. Rotate the adjacent clearants at the same time. Whenever the appearance of a solution looks unusual (e.g. milky alcohols, appearance of beads of water or clearant), discard and replace that solution immediately. Stain exchange Use the information provided by the manufacturer along with microscopic examination of control slides, it will help determine a regular replacement schedule. Bluing Reagent Prepared bluing reagents should be changed at least daily. 9

10 Tissue-Tek Film When coverslipping with Film: ALWAYS USE XYLENE AND PERFORM GOOD DEHYDRATION!

Tissue-Tek Film Removal of coverslipping film (see operating manual section 3.1 for more detailed information) 1. Place the slides in acetone until film can be peeled off (approx.

11 Tissue-Tek Film Removal of coverslipping film (see operating manual section 3.1 for more detailed information) 1. Place the slides in acetone until film can be peeled off (approx. 5 min. to 10 min.). 2. Place the slides in clean acetone for 5 min. 3. Place the slides in ethanol 100%. 4. Place the slides in xylene. 5. Slides are now ready for further treatment. 11

12 Staining spectrum Routine H&E DNA-RNA: In situ hybridization Staining Special stains IHC

13 What is optimal staining quality? Desired end results of a stain is often based on individual preferences: Section thickness Intensity of stain Shade of stain The choice of reagents are also a main factor affecting the end results of a stain: Commercially prepared or in-house prepared Availability and quality Safety and cost No matter what preferences, the staining results should always be reproducible and of good quality.

14 Variables affecting the staining quality Fresh tissue Grossing Fixation Decalcification Cryotomy Glass slide Waterbath Microtomy Embedding Processing Staining Cover slipping Microscopy

15 Dewaxing: common problems Residual wax cause staining faults - may be obvious or more subtle: Decreased colour intensity Irregular staining Wax removal is slower when slides are cold and when sections are thicker: Make use of the oven Different solvents remove wax at different rates: Use at least 2-3 stations of Xylene or Xylene substitute, 3 minutes per station Xylene substitutes are slower and often require longer time or more stations compared to Xylene All solvents are less efficient when they contain excessive wax: Make sure to check upon and change the solvents regularly

16 Dewaxing: consequences Blue spots or unstained areas Wax residuals in sections Use at least 2-3 stations of Xylene or Xylene substitute, 3 minutes per station, consider the configuration and use the oven

17 Alcohols: common problems Used both when rehydrating (adding water) and dehydrating (removing water) tissue sections. The grade and formulation of alcohol is important: Fading of the stain or poor staining results due to presence of denaturants Hazy or milky appearance due to poor dehydration Immersion times and stations should be sufficient to assure complete removal of the previous solutions and water: Use at least 2-3 stations of each alcohol when possible, 1 minute per station Alcohols are less efficient when they are exhausted: Make sure to check upon and change the alcohols regularly Rotate alcohols frequently, especially during humid conditions

18 Alcohols: consequences Muddy staining Water retained in the tissue section due to overused alcohols Reagent should be changed Make use of the solution exchange reminder

19 Other common problems and consequences Weak & uneven staining Improper washing step leaving residual alkali from bluing reagent hindering eosin staining Alkali residuals need to be removed by washing Make sure to add a water wash step in the staining program

Other common problems and consequences Weak staining Alcohol rinse too long, aqueous or excessive ph too high Inadequate staining time ph is a critical

20 Other common problems and consequences Weak staining Alcohol rinse too long, aqueous or excessive ph too high Inadequate staining time ph is a critical element which needs to be controlled (staining solutions, replacement rate, water acidity) Check programmed staining time and reagent replacement schedule 20

21 Other common problems and consequences Sections partially dried and tiny air bubbles are trapped (black colorization) Section allowed to dry out before coverslipping Continously remove slides from PE stations and make sure End stations are filled properly with solvent

22 Troubleshooting How to analyze the issue What kind of artefact is seen in the sample? How many samples are affected? (only one block, the hole run, only one case, all runs) Was the correct staining protocol used? Check the reagents, visually and by expiry date Is it likely that the program was too short or too long for these samples? How is the thickness of the sections? Did the instrument give any errors?

23 Troubleshooting If you are still not sure about the problem Examine your specimens and collect data. Document your experiences (how often, which tissue types etc) maybe you see a pattern. Always feel free to contact us and pictures make it much easier to understand the issue.

Techniques and their diagnostic application Diagnostic Histochemistry

24 Literature Histological & Histochemical Methods: Theory and Practice Theory and Practice of Histological Techniques Manual of Histological Techniques and their diagnostic application Diagnostic Histochemistry By: J A Kiernan By: John D Bancroft By: John D Bancroft Harry C.Cook By: Mark Wick

25 Thank you!

: In order to study tissues with a microscope they must be preserved (fixed)- fixation Following fixation, blocks of tissue must be cut into thin

: In order to study tissues with a microscope they must be preserved (fixed)- fixation Following fixation, blocks of tissue must be cut into thin sections.-microtomy Other techniques involve dehydration