Hallo Maryam,

try to cool down your paraffin blocks by applying ice directly on them just before using the microtome. You can also use warm water in order to spread the paraffin section after being cut by the microtome.

Hi Maryam,

this is how we process brain tissue:

Preprocessing is comparable to your protocol, except that we only fix for 24 h (4 % formalin) before dehydration. However, I don't expect this to make a huge difference.

For microtome cutting (3 µm sections) the paraffin blocks are placed on a cooling plate at least 15 min before starting and during all the breaks.

The cut sections are transfered to 30 % Ethanol/ H2O where we pick them up with the glass slides. Then the individual slides are *quickly* dipped in a water bath of 45 - 55 °C in an (almost) perpendicular way until the wrinkles are gone. During this, the paraffin cuts will partly detach from the glass slide and float on the water.

This step is very crucial, because the tissue tears if it takes too long or the water bath is too hot. If the water bath is too cold and/or you don't wait long enough, there will be residual wrinkles. It is very recommendable to do this carefully near the edge of the waterbath since the cuts tend to float away. We have a tiny waterbath for this purpose only.

Best test some different angles/ water bath temperatures. In my experience, temperature is the most decisive factor.

Afterwards, cuts are dried over night at 37 °C. According to an old histology book in our lab, it is also possible to dry 1 h at 60 °C or 2 days at RT. Haven't tried it though.

This way, our cuts are relatively flat. However, some wrinkles/ fissures can't be avoided in my experience (for my important samples, a very experienced technical assistant does the cutting and even she can't avoid it completely).

Good luck!