Tips / FAQ's
While every reagent assembly kit must be treated individually, there are common points which apply to all .The following items are answers to some commonly asked questions. Of course, if you have any other questions please feel free to take advantage of Poly Scientific’s technical assistance department.
- In general, an 8oz. kit will stain 100 slides and a 16oz. kit will stain 200.
- All timings given are representative and should be adapted to your particular laboratory conditions.
- All silver stains should be performed in acid cleaned glassware for best results.
- Use only deionized or distilled water, unless otherwise directed.
- Most working solutions, once made, should be used immediately.
- All solutions are individually labeled with expiration dates and storage conditions.
- All procedures are written for manual use, but all can and have been adapted for any automated stainer.
FIXATION TIPS
- Tissue should be fixed immediately after removal from the body.
- Tissue should be cut to less than 5 mm in thickness for best
results. - The volume of fixative should be 10-20 times the tissues.
- Formalin penetrates most tissue at a rate of 0.5mm per hour.
- Heat, agitation and vacuum will all speed up the rate of
penetration of formalin. - Always use high quality reagents to maximize performance.
- 10% Buffered Formalin should be used for long term tissue storage.
PROCESSING TIPS
- Tissue should be no larger than 5mm in thickness.
- Always take into account the thickness of your tissue
when determining station times. - Avoid heat during dehydrating and clearing to reduce
the possibility of overprocessing. - The use of vacuum is always encouraged during
processing. - Establish a reagent rotation schedule suited to your lab.
- Always use high quality reagents to maximize
performance. - A hydrometer is a practical, easy way to determine when to rotate or replace alcohols.
- Use Xylene, not a substitue, in the cleaning cycle.
Troubleshooting H & E
| Problem | Cause | Solution |
|---|---|---|
| Dull Uneven Hematoxylin Staining | Improper Fixation | - Increase time in fixation and maintain an appropriate schedule |
| Overdifferentation | - reduced the time in differentation to minimum neccessary | |
| Depleated Hematoxylin | -determine and maintain a proper rotation schedule | |
| Incomplete Deparaffinization | - use three stations of xylene and maintain a rotation schedule | |
| Dull Uneven Eosin Y Staining | Improper Fixation | - increase time in fixative and maintain an appropriate schedule |
| Depleated Eosin Y | - determine and maintain a proper rotation schedule | |
| Solution Carryover | - drain slides thoroughly inbetween stations to prevent carryover | |
| Bleeding | - rotate final xylenes to prevent carryover of alcohol into coverslips | |
| Cloudy Murky slides | Incomplete clearing | - determine and maintain a proper rotation schedule and use three stations of xylene |