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Histopathology

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mazharul_vet:
Dear All
Good morning.
I have previous experience to work with NBF fixed paraffin blocked sectioning and staining. I have joined in a new laboratory where cryo-sectioning (with THERMO; HM525) is practiced.
The protocol is –
1. Fixing the tissue in 10% formalin (not buffered) for 24 hrs.
2. Trimming and washing with running tap water for 1 hr.
3. Putting it on stage for freezing.
4. at -20°C (all tissue): sectioning the tissue
5. Incubation of the slide containing sectioned tissue at 37°C
6. Hematoxylin for 20 minutes
7. Washing with running tap water for 10 minutes
8. Eosin for 2 minutes
9. 50%, 75%, 80%. 90%, 95%, absolute, absolute, xylene, xylene for 2 minute each
10. Mounting

This protocol is being practiced for long years and they were getting result.

The technician went on vacation; I am trying to do histopathology but failed. My sections do not come properly. I do not get appropriate structure of the tissues. Even the Eosin is not stained.

There is no body here to help me. Can anybody guide me in this regard.

Thanking you

Md. Mazharul Islam

excalibur:
Lots of unneccessary steps and hemat staining times can be changed to speed things up.

1 hour rinse in not needed.

Cryoprotecting in sucrose? Are you placing the tissue in OCT medium prior to freezing? Is the wet tissue frozen without blotting excess water? You may be getting ice crystal formation.

Air dry slides 30 min.

Hemat stain 10 min.
Rinse in water 2X.
Blue in 1% ammonium hydroxide ~ 30 sec.
Rinse in water 2-3X.

What eosin solution is used? Skip the 50, 75, and 80% EtOH and add another 100% EtOH and xylene.

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