10% Normal Buffered Formalin


1. For Animal


  1. needle connected tubing
  2. peristaltic pump
  3. Scissor
  4. forceps
  5. Scalpel
  6. 1x PBS (can be made from 10X PBS; Cat# PBS01-32R)
  7. 10% neutral buffered formalin (Cat#NBF07-32R)
  8. Anesthesia (phenobarbital etc.)



  1. Anesthetized animal.
  2. Cut the skin below the sternum and open the thoracic cavity and inserted the needle which connected the tubing that has PBS onto apex of the heart through the aorta and cut the right atrium to let the blood out.
  3. Or you can put the tubing into the femoral artery and cut the femoral vein to let the blood out.
  4. Run 1xPBS (approximately 50-60mL) through the animal slow speed to wash the cardiovascular system from the blood.
  5. Once the blood appears translucent, change the PBS with 10% NBF and run with the medium speed (1-2 ml/minutes) to match hearth rate of the animal.
  6. Perfuse the animal adequately at least 1ml for 1 gram body weight (if you have animals weighted 300 gr, you need to perfused the animal with 300 ml of NFB).
  7. Make sure the animal is stiffed post-perfusion, if not you need to correct the position of your needle.



2. For Fresh Tissue

Cut the tissue not larger than 1cm3 to get better penetration into the cells


3. For long storage/prevation

Put your object in the container (can be plastic or glass) contained 10% NBF and close it thigh.

https://tissueprotech.com/img/cms/10% NFB_1.pdf




Anti freeze solution (AFS) is a preservative solution to protect the morphology and antigenicity of the fixed tissue. The sample storage in AFS are stable indefinitely in -20 °C. The solution is suitable for various fixed samples from animal and human tissue. The morphology and quality of immunostaining of the samples are never change.




Animal and human tissue: the fixed tissue must be cryoprotected (put in gradient sucrose 10% to 30% until they sunk) and cut in cryostat or freezing microtome.  Put them in the multidisc/well contained AFS. You may collect them as serial section for quantification purposes or multiple histology/immunostaining. Keep them in -20 °C for indefinite time.


Sample storage:

After submersing in AFS, the sample can be stored –indefinitely -20 °C. Sample need to wash with buffer solution prior their staining.



(CAT# CVA06-500R)



  1.      0.1% Cresyl Violet Acetat solution(Cat# CVA06-500R)
  2.      Differentiation solution: 2 drops glacial acetic acid in 95% alcohol.
  3.      Gradient Alcohol (70%, 95%,100%)
  4.      Distilled and Tap water
  5.      Xylene or HistoClear




  1.         Dewax sections in Xylene or HistoClear (2-3 change of 3 minutes each)
  2.         Rehydrate in alcohol (100%, 95%, 70%), 3 minutes each
  3.         Rinse in tap water
  4.         Stain in 0.1% Cresyl-Violet 4-15 min
  5.         Quick rinse in tap water to remove excess stain
  6.         Dehydrate in alcohol (70%, 95%,)3 minutes each
  7.         If required immerse sections for 2min in Differentiation solution – check staining on microscope.
  8.         Dehydrate in absolute ethanol (100%, 2 change of 3 minutes each)
  9.         Clear the staining with Xylene or HistoClear and mount.
  10.      Allow dry in fume hood

https://tissueprotech.com/img/cms/CV staining protocol.pdf


(CAT# HE09-40R)



  1.      HE stain kit (Cat# HE09-40R) contains:

-       Hematoxylin solution (Cat#H08-20R)

-       Eosin Y solution (Cat#07-20R)

  1.      0.3% hydrochloric acid alcohol.
  2.      Gradient Alcohol (70 -100%)
  3.      Distilled and Tap water
  4.      Xylene or HistoClear




  1.         Dewax sections in Xylene or HistoClear (2-3 change of 3 minutes each)
  2.         Rehydrate in alcohol (100%, 95%, 70%), 3 minutes each
  3.         Bring sections to distilled water 
  4.         Stain nuclei with the haematoxylin (Cat#H08-20R) for 4-10 minutes
  5.         Rinse in running tap water
  6.         Differentiate with 0.3% acid alcohol
  7.         Rinse in tap water
  8.         Stain with eosin (Cat#07-20R) for 30 second - 2 minutes
  9.         Dehydrate in alcohol (70%, 95%, 100%), 3 minutes each
  10.      Clear the staining with Xylene or HistoClear and mount.
  11.      Allow dry in fume hood

https://tissueprotech.com/img/cms/H&E staining protocol.pdf