Ab8878

The following antibodies were applied: Tetraspanin-4 (NBP1-59438, Novus); RPE 65 (MA1-16578; Thermo Fisher Scientific); GFAP (ab279290, Abcam); CD11b (ab8878, Abcam); integrin α5 (ab288767, Abcam); EOGT (ab190693, Abcam); NDST1 (26203-1-AP, Proteintech); Alix (2171, Cell Signaling); Alix (92,880, Cell Signaling); Smad2/3 (3102, Cell Signaling); Phospho-Smad2/Smad3 (8828, Cell Signaling); Alpha-Smooth Muscle Actin (MA5-11547, Thermo Fisher Scientific); GAPDH (5174, Cell Signaling) and β-actin Rabbit antibodies (ab8227, Abcam). Secondary antibodies used included the following: Alexa Fluor 488 Anti-Mouse; Alexa Fluor 555 Anti-Rabbit; Alexa Fluor 555 Anti-Mouse; and Alexa Fluor 555 Anti-Rabbit (Thermo Fisher Scientific). Additionally, CCK8 (ab228551, Abcam), Dulbecco’s modified Eagle’s medium (DMEM)/F12 culture media, and fetal bovine serum were obtained from Thermo Fisher Scientific.

Mice were transcardially perfused with 20 ml PBS. Brains were fixed overnight in 4% paraformaldehyde in PBS at 4°C and processed for frozen sectioning as before [43 (link)]. A coronal rodent brain matrix (RBM-2000C, ASI Instruments) was used to obtain consistent blocks of pontine regions that included both facial nuclei. Cryosections (14-μm) were collected from the entire facial nuclei on coated glass slides and stored at − 20°C until use. Tissues were permeabilized in blocking solution (0.1% Triton-X 100, 5% bovine albumin, normal goat or normal donkey serum, and PBS) for 1 h at room temperature and incubated overnight at 4°C with primary antibodies: 1:200 rat anti-CD11b (ab8878, Abcam), 1:500 rabbit anti-IBA-1 (019–19,741, Wako), 1:200 goat anti-APOE (AB947, Merck), and 1:500 rabbit anti-CCL5 (RANTES) (710,001, ThermoFisher Scientific). Antigen retrieval was performed at 96 °C prior to APOE staining for 40 min in 10 mM citrate buffer at pH 9. Sections were incubated with corresponding secondary antibodies conjugated to 1:1000 Alexa Fluor 488 or Alexa Fluor 647 (Life Technologies) and 1:5000 nuclear counterstain 4′,6-diamidino-2-phenylindole (DAPI, Sigma) for 2 h at room temperature, and mounted in ProLong® Diamond Antifade Mountant (Life Technologies).

The eyes were harvested on the 7th day after alkali injury and embedded in optimal cutting temperature (OCT) compound after the fixation with 4% paraformaldehyde overnight at 4°C. We blocked 5 μm OCT frozen sections with 3% BSA for 1 h at room temperature and incubated the sections overnight at 4°C with a rat anti-F4/80 antibody (Abcam, ab66440, 1:200), rat anti-CD11b antibody (Abcam, ab8878, 1:200) or rabbit anti-CD31 antibody (Abcam, ab28364, 1:100). Detection of bound anti-F4/80, anti-CD11b or anti-CD31 antibodies was performed using anti-rat IgG Alexa Fluor 488 (Cell Signaling Technology, Germany, 1:1000), anti-rat IgG Alexa Fluor 555 (Cell Signaling Technology, Germany, 1:1000) or anti-rabbit IgG Alexa Fluor 555 (Cell Signaling Technology, Germany, 1:1000). After counterstaining with DAPI (Abcam, ab228549), the sections were mounted with antifade mounting medium (Beyotime, Shanghai, China). The sections were viewed and photographed with fluorescence microscopy (DMI3000 B; Leica, Wetzlar, Germany).

For the corneal inflammatory cell recruitment test, mouse eyes were embedded in a compound at the optimal cutting temperature, sequentially sectioned (8 µm) and stored at −80°C. Subsequently, the frozen sections were blocked with 3% bovine serum albumin at 37°C for 1 h. Then at 4°C, the frozen sections were inducible rat anti-F4/80 (Abcam, ab66440, 1:100) and rat anti-CD11b (Abcam, ab8878, 1:100) overnight. Sections were then washed with phosphate buffered saline and incubated with anti-rat IgG Alexa Fluor 555 (cell signaling technology, 1:1,000) and anti-rat IgG Alexa Fluor 488 (Cell signaling Technology, Germany, 1:1,000) for 1 h at room temperature, followed by DAPI (Abcam, Ab228549) solution staining. Photographs were taken with a fluorescence microscope (DM 4000B). The number of positive cells was quantified using Adobe Photoshop CC (Adobe Systems, Inc., San Jose, CA, United States) with the method described by et al. (Dai et al., 2018 (link)). All histological assessments were performed as blinded studies by the same two observers.