To identify the limbal niche cells or LNCs by immunofluorescence staining, add 1 milliliter of 0.25%trypsin EDTA per well to the LNCs cultured in a 5%matrigel coated 6 well plate. Digest the cells by incubating the plate for five minutes at 37 degrees Celsius. Quench the digestion by adding 1 milliliter of knockout serum containing MESCM to the cell suspension.
Transfer the cell suspension to a centrifuge tube and centrifuge it 200G for five minutes before carefully aspirating the supernatant. Re-suspend the pellet in one milliliter of MESCM. Next, using a cytofuge, deposit 80 microliters of the cell suspension equally on four microscope slides per the manufacturer's instruction.
Fix the cells with 4%paraformaldehyde for approximately 10 minutes. Then permeabilize the cells on the slides using 0.2%Triton X-100 in PBS for 15 minutes. Block the cells with 2%bovine serum albumin for one hour before incubating them with primary antibodies on a shaker overnight at 4 degrees Celsius.
Post incubation, remove unbound antibodies by washing the slides with PBST three times for five minutes each. Next, incubate the sample with suitable secondary antibodies at 37 degrees Celsius for one hour before washing any unbound antibodies as demonstrated previously. Counterstain the nuclei with DAPI having a concentration of 5 micrograms per milliliter.
After sealing the slides, perform imaging using a fluorescence microscope. Using an RNA isolation kit extract the total RNA from the LNCs per the manufacturer's instructions. Then using a high capacity complementary DNA or CDNA transcription kit, reverse transcribe 1 to 2 micrograms of the RNA.
Prepare a 20 microliter solution containing 2.5 microliters of the CDNA, 0.8 microliters of corresponding genetic primer, 10 microliters of a universal PCR master mix, and 6.7 microliters of double distilled water. Then perform a quantitative polymerase chain reaction using the appropriate conditions. Finally, employ the comparative CT technique to examine the relative gene expression.
Double immuno staining of the fourth passage LNCs clearly revealed that these cells were consistently panCK negative, VIM positive, CD90 positive, CD105 positive, SCF positive, and PDGFR positive.
