Hello Nazmul Islam,

Microtome sections can be difficult to perform depending on different parameters:

• The fixation time of the tissue (if it is overfixed, the tissue will be fragile and "breakable" for example). Here, the fixing time seems to me correct considering the size of the tissue.

• The age of the blade which cuts the block (it is sometimes necessary to have a recent blade but also of a well defined nature for certain tissues)

• The nature of the paraffin (some tissues require a special high quality paraffin).

• The ambient temperature & of the block (the tissue sample and the paraffin are cut much better when they are cooled on a plate provided for this purpose or beforehand with ice cubes). In this month of July it may be too hot in your lab...

Hoping that will be useful for your spleen sample analyses.

Ludovic Dumont, Ph.D.

Hi Ludovic Dumont , thank you very much for your response. I want to know how much fold of fixation ( how much volume of fixation per spleen sample) we should use?

After embedding do we need to keep the spleen sample some hours or overnight in -20 or 4 degrees before sectioning the tissue with microtome?

Hello Nazmul Islam,

Fixatives are poor buffers; therefore, the pH of solution tends to change during the process of fixation. A large volume of fixative will assure an optimal fixation.

The volume of fixative used should be approximately 15 to 20 × the volume of the tissue. A large volume will tend to perform fixation adequately while keeping the reagent stable.

For best results, tissue should be cut to into pieces that are no more than 4 to 5 mm thick in one dimension. If the tissue of interest is needed for other purposes/applications (molecular tests, etc.), it can be cut and snap-frozen separately before fixation.

For Paraformaldehyde solution, it is important to use freshly prepared PFA 4% solution at 4°C (or, at least, from a 16% stock solution stoked at -20°C for a reduce amount of time).

Hoping that will be useful for your spleen sample PFA 4% fixation.

Ludovic Dumont, Ph.D.

Hello Nazmul,

I applied one of the following techniques to any bloody tissue.

In my experience, I suggest 2 methods:

1. After trimming and exposing the tissue, fine trim to make the surface evenly trimmed, then I soak in decal solution for 10 minutes , rinse the block in water, chill in icy water, then cut.

2. Another method, after trimming soak in 5% ammonia water, chill in icy water , then cut. Note: After soaking in ammonia water , do not oversoak in chilled water nor ammonia water .

Good luck.

Ludovic Dumont thank you for your response, according to your suggestions I used a large volume of fixation and it worked properly. Thank you.

Lilian Antonio Thank you for response