Tissue Fixation and Freezing

 

1. Place PBS perfused tissue in 2% paraformaldehyde for 1-2 hours. The tissue needs to be submerged in the solution. Larger tissues need 2 hours incubation in 2% paraformaldehyde (lobe of liver). One hour is sufficient for a small tissue such as lymph node. 

2. Place tissue in 30% sucrose for 24 hours having changed the sucrose 2-3x within 24 hours. Provide enough 30% sucrose to submerge the tissue. 

3. Label a cryosample bag with the following information: tissue type, Sample ID #, Experiment Date. 

4. Cryopreservation occurs with placing the tissue in a metal dish containing 2-Methylbutane (Fisher CatNo. O3551-4). This metal dish of 2-Methylbutatne is placed in a Styrofoam box containing liquid nitrogen. 

5. Once the sample has been slowly frozen, place the tissue in the labeled cryosample bag that is already cold (too minimize thawing of the tissue). Store tissue at -80C until ready for sectioning.