Primary antibodies: mouse anti-GFAP (1:1000) and rabbit anti-TIII (1:1000). and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation MG149 protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures. Keywords:Neuroscience, Issue 45, cryopreservation, neuron, astrocyte, neuronal stem cells, tissue culture, brain cortex Download video stream. == Protocol == == 1. Cryopreservation of Cortical Tissue Blocks == == Material Preparation == Prepare 1X Hank’s Buffered Salt Solution (HBSS) by diluting at 10X stock solution in sterile water (1:9). Store HBSS at 4oC. Prepare 10% DMSO freezing medium by diluting DMSO in HBSS (1:9). The freezing medium should be made fresh before cryopreservation and stored at 4oC until chilled. A steel razor blade is used for the systematic chopping of tissue. Before use, the razor blade is usually sterilized by submersion in 70% ethanol for 2 hr. Immediately before tissue processing, rinse the razor blade with sterile water 3 times. Avoid leaving the razor in sterile water for an excessive amount of time, as it is prone to oxidation. A Nalgene freezing container is loaded with cryovials (2.5 mL) and then brought into a sterile biological cabinet. 1 mL of chilled freezing medium is added to each vial. The freezing container is usually then placed at 4oC for at least 2 hrs. == Cleaning, Chopping, and Freezing == The brain tissue to be frozen is cleaned of meningeal membrane and blood vessels. Use sterile needle tips to carefully remove debris from the tissue while working on top of an ice pack. Cleaned tissue should be rinsed lightly with cold HBSS and transferred to a new Petri dish for chopping. Working on top of an ice pack, use a sterilized razor MG149 blade to rapidly chop the tissue into approximately 1 mm3blocks. Working with small portions of cleaned tissue at a time results in MG149 better control over the chopping procedure, resulting in more uniform block Serpinf2 sizes. Slowly add chopped tissue into 50 mL of HBSS in a 50 mL conical tube. Gently rinse the Petri dish with HBSS in order to collect any remaining tissue blocks. Allow the tissue blocks to descend to the bottom of the tube, creating a loosely packed tissue block pellet. While the tissue is settling, bring the previously chilled freezing container and cryovials into the biological safety cabinet. Uncap all cryovials to hasten the process of adding tissue. After all the tissue blocks have settled, gently aspirate the excess HBSS, leaving a very thin layer of media above the pellet. Centrifugation is usually discouraged as it will cause the tissue blocks to adhere to each other, significantly diminishing freezing efficiency. Slowly collect 200 L from the bottom of the loose tissue block pellet by using pipette tip with wide orifices (cut tip with sterile scissors). Transfer the material to one cryovial and move on to the next, again collecting tissue from the bottom of the pellet. It is essential that the entire tissue allocation procedure does not take more than 3-4 min per freezing container. This ensures that the tissue is exposed to DMSO for only a short period of time before freezing. After transferring the tissue to the vials, place the freezing container in a -80oC freezer for at least 4 hrs. Alternatively, the freezing container can be left overnight at -80 oC. Repeat this process for any remaining freezing containers. Transfer the cryovials from the freezing container(s) to a cryobox and place in a liquid nitrogen tank for long-term storage. == 2. Thawing and Culturing of Frozen Cortical Tissue Blocks == == Material Preparation == One day prior to thawing tissue samples, poly-L-lysine coated Petri dishes/coverslips are prepared. In general, for cortical neurons MG149 we prepare coated dishes with 500 g/ mL or 1mg/ mL. Add enough poly-L-lysine solution (made in borate buffer) to fully cover the bottom of the dish. If.