A11033

We performed CIC on the subjects' superior tarsal conjunctiva. The CIC samples were collected at the center of the superior tarsal conjunctiva. In preparation for CIC, subjects underwent local anesthesia with 0.4% oxybuprocaine hydrochloride eye drops. Subsequently, a sterile membrane (0.45 μm, Millipore, Boston, MA) was placed on the surface of the palpebral conjunctiva and pressed for a few seconds. To increase the number of collected cells, the palpebral conjunctiva was gently wiped with cotton swabs to keep it dry.
Indirect immunofluorescence was used to observe the staining of CD4⁺ and CD8⁺ cells in CIC samples. After fixing the CIC sample with 4% paraformaldehyde, antibodies against CD4 (A0362, 1:200, Abclonal, Wuhan, China) or CD8 (A11033, 1:200, Abclonal) were incubated at 4°C overnight. After washing, we stained CD4 with an Alexa Fluor 488-conjugated anti-rabbit antibody (A-11034, 1:1000, Invitrogen, Shanghai, China) or CD8 with an Alexa Fluor 594-conjugated anti-rabbit antibody (A-11037, 1:1000, Invitrogen) at room temperature for 50 minutes. The cells were observed with a Leica fluorescence microscope (LEICA DMi8, Leica Microsystems, Wetzlar, Germany). Three different fields of each sample were observed and evaluated to count the positively stained cells.

After subjects received adequate topical anesthesia with oxybuprocaine hydrochloride eye drops, sterile membrane (0.45 µm; Millipore, Boston, MA, USA) was placed on the surface of the superior tarsal conjunctiva, gently pressed for 5 seconds, and then removed, and stored at −80°C.
Filter membranes were fixed with 4% paraformaldehyde for 1 hour. After washing with phosphate-buffered saline, filters were blocked with 0.1% Triton at room temperature for 20 minutes, and incubated with anti-CD4 antibody (A0362, 1:200; Abclonal, Wuhan, China) or anti-CD8 antibody (A11033, 1:200; Abclonal) at 4°C overnight, followed by incubation with Alexa Flour 488 anti-rabbit secondary antibody (A-11034, 1:1000; Invitrogen, Shanghai, China) for CD4 or Alexa Flour 594 anti-rabbit secondary antibody (A-11037, 1:1000; Invitrogen) for CD8 for 1 hour at room temperature. Stained samples were examined and photographed with a fluorescence microscope (LEICA DMi8; Leica Microsystems, Wetzlar, Germany) at 400 times magnification. Three fields of view for each sample were randomly chosen, and images were analyzed manually using Image J software to count DAPI⁺ cells (blue, total cell number), CD4⁺ cells (green), and CD8+ cells (red). Then, the percentage of CD4⁺ DAPI⁺ cells and CD8⁺ DAPI⁺ cells was calculated for subsequent statistical analysis.