iam in need to know what is the best fixative and its preparation protocol to fix fish muscles and liver for latter histopathology ?
Dear Afaf Mohamed ,
I usually use the Bouin's fluid (Aquous solution of picric acid = 75ml + formaldehide PA = 25ml + 05 ml de Acetic Acid) for animal tissues in general, for traditional histology with hematoxylin-eosin .
This is because it does not deform the histological parts.
It causes immediate cristalization of proteins, which prevents the muscle fibers to contract with the fixation .
It also allows a brighter color, leaving the affinity heads anilines more receptive to the stainings.
It is a faster fixator and the histological fragments should have a maximum of 1 cubic centimeter. Use of 8 to 20 times the volume of the histological fragment by up to 3 days.
Empty Bouin liquid and pass the pieces to 70 % alcohol.
But if you are studying glycogen storage , I indicate the alcoholic Bouin ( Gendre ) , because the alcohol will not allow the muscle or liver glycogen dissolves and colorings with Periodic Acid Schiff occur properly .
Kind regards.
Hi, I have not worked with fish tissues but I have experience with muscle tissue from rats even with muscle cell culture and in both cases the paraformaldehide 3% or 4% works well, in tissue, I apply the PFA for 30 minuts. I think that the fish muscle and rat muscle can not be much different, maybe this reagent is usefull for you. If you want I have a protocol to prepare PFA.
Best whishes!
Dear Afaf Mohamed ,
I usually use the Bouin's fluid (Aquous solution of picric acid = 75ml + formaldehide PA = 25ml + 05 ml de Acetic Acid) for animal tissues in general, for traditional histology with hematoxylin-eosin .
This is because it does not deform the histological parts.
It causes immediate cristalization of proteins, which prevents the muscle fibers to contract with the fixation .
It also allows a brighter color, leaving the affinity heads anilines more receptive to the stainings.
It is a faster fixator and the histological fragments should have a maximum of 1 cubic centimeter. Use of 8 to 20 times the volume of the histological fragment by up to 3 days.
Empty Bouin liquid and pass the pieces to 70 % alcohol.
But if you are studying glycogen storage , I indicate the alcoholic Bouin ( Gendre ) , because the alcohol will not allow the muscle or liver glycogen dissolves and colorings with Periodic Acid Schiff occur properly .
Kind regards.
I'm agree with Renato Zacarias Silva. And also if you need to fix the fish in whole you can put the live fish directly to the Bouin's fluid (if the fish is small). Later you can see well fixed internal organs such as intestine, liver and stomach. After put in Bouin's fluid you can remove the extra yellow color caused by the picric acid, put them in a alcohol series day by day.
best regards.
Hi,
I'm using 10% neutral phosphate buffered formalin (pH=7.2) for fish samples. It works well.
For preparation of 1000 ml NPBF, please add 100 ml formalin solution (37%) to 900 ml distilled water. Then add 6.5 g Na2HPO4+ 3g NaH2PO4 to your solution. Now, it's ready to use. It takes a few days or even a week to fix your tissue samples.
Note: Phosphate Buffer preparation is dependent on your desired pH and water molecules content in your mono/dibasic sodium phosphate salt, please check it before adding.
Note: Please renew your fixative after 24 h!
Bouin's Fixative is an alternative option. The exact protocol was described above by Renato.
Good luck,
thanks a lot for all for your answers
Pedram Malekpouri if you please when i have to check ph before adding 6.5 g Na2HPO4+ 3g NaH2PO4 or after ?
thanks in advance
Check your phosphate salt for H2O molecules initially and check your final solution pH using a pH meter in the end.
The choice of fixative solution depends on aim of your research work. If you need only observation slides then all common fixatives are suitable as for example:BNF(10% buffered neutral formalin),Bouin's or Helly's fixatives et cet.
Dear Afaf,
An important practical tip is to ensure that the fish is freshly euthanased and to have the muscle and liver portions promptly dissected out (< 5 minutes post death), and not exceed 1 cubic cm size of tissue, to place into 10% NBF in a ratio of 1 : 10 volume of tissue to fixative. Making scalpel slices into the muscle or liver to encourage rapid penetration of the formalin fixative is useful for larger pieces which may contain the lesion of interest. This will prevent the risk of artefacts leading to suboptimal fixation and issues with proper interpretation. Check out the EAFP fish necropsy manual for further guidance.
Cheers
Roger
http://necropsymanual.net/en/
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