Hi,
I need to nissl stain some California Killifish brains.
The brains have been dissected out and placed in 4% paraformaldehyde (made With 0,1M sørensensbuffer) over night, then they were washed and placed in sucrose over night, before they were frozen at -80oC.
The first staining went very poorly, and the sections ended up looking cracked, dry and white.
After sectoning on a cryostat set at -20oC, and With 100um thick sections I put the slides in the oven at 65oC for 5 min before freezing them at -80. The day after I again placed them in the oven at 65oC for 5 min before starting the staining protocol.
The protocol is as follows:
1 min in dH2O
10min in crecyl violet
1min in dH2O
2 min in 70% EtOH
2min in 80% EtOH
2 min in 90% EtOh
2 min in 96% EtOH + Acetic Acid
2 min in 100 % EtOH
2 min in xylene
After this they are covered With a coverslip and let to airdry over night.
Thanks in advance,
Siri
Hi Siri, all this recommandations you will find in my attached Cresyviolett-staining protocol. It's suitibal for frozen sections as well as for paraffin sections. In Chinmaya' s and im my protocol we start with descending alcohol with or without xylene. This is important because brain tissue is a very fatty tissue and CV is a watersoluble dye. To get deep in to the sections it is nacessary to remove all the fat from the tissue. This might be also an explication why your staining result is so pale.
To store mounted sections in the -80°C is one possibility. If you are working with thick sections that means 30µm and thicker I recomand to store them unmounted in a solution containing 30% sucrose and glycerol (a/a) in kryovials at -20°C. You can store this sections for sevaral years. The stored sections have to be rinsed in PBS or TBS several times before starting with CV-staining or Immunohistochemistry or what ever you like to do. Good luck and best regards, Ute
Thank you. How is your samples treated before the staining?
Hi Siri, all this recommandations you will find in my attached Cresyviolett-staining protocol. It's suitibal for frozen sections as well as for paraffin sections. In Chinmaya' s and im my protocol we start with descending alcohol with or without xylene. This is important because brain tissue is a very fatty tissue and CV is a watersoluble dye. To get deep in to the sections it is nacessary to remove all the fat from the tissue. This might be also an explication why your staining result is so pale.
To store mounted sections in the -80°C is one possibility. If you are working with thick sections that means 30µm and thicker I recomand to store them unmounted in a solution containing 30% sucrose and glycerol (a/a) in kryovials at -20°C. You can store this sections for sevaral years. The stored sections have to be rinsed in PBS or TBS several times before starting with CV-staining or Immunohistochemistry or what ever you like to do. Good luck and best regards, Ute
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining