Fixing and Blocking 2D Human Umbilical Vein Endothelial Cell (HUVEC) Cultures for Immunohistochemical Analysis

To begin, add 30 milliliters of 5%potassium hydroxide in methanol to a 50 milliliter tube. Incubate 100 cover slips with a diameter of 12 millimeters in the tube for 30 minutes under constant agitation. Next, wash the cover slips in demineralized water for 30 minutes.

Once washing is complete, transfer the cover slips into 70%isopropyl solution for storage. To coat the cover slips, first lean them individually against the wall of a square Petri dish. When the isopropyl alcohol has evaporated, transfer them into a 24 well plate.

Pipette one milliliter of collagen in PBS into each well and incubate. Next, wash the cover slips about 4 to 5 times for five minutes each with one milliliter of PBS. Pipette cultured HUVECs into the wells with the coated cover slips.

After culturing the cells for a desired length of time, wash the cells in one milliliter of PBS for five minutes. Fix the cells in one milliliter of 2%paraformaldehyde for 20 minutes at room temperature. Now, rinse the cells in one milliliter of PBS for 3 to 4 wash cycles of five minutes each.

Finally, pipette one milliliter of blocking buffer into the well plate before staining.