Permaflour

Eight days after adoptive transfer and infection one third of the spleens was fixated in 4% formaldehyde for two hours, dehydrated in 30% sucrose for 24 hours and frozen with liquid nitrogen in Tissue-tek O.C.T. compound (Sakura Finetek Europe; Alphen aan den Rijn, Netherlands). On glass slides cryostat sections of 7 μm thickness were blocked with 5% BSA (Sigma-Aldrich) for 30 minutes, followed by staining with PE conjugated CD90.1 antibody diluted 1:500, FITC conjugated CD45R/B220 antibody and APC conjugated CD4 antibody both diluted 1:200 in 5% BSA, containing DAPI (all from BioLegend) diluted 1:8000. After one hour the slides were washed with PBS three times for three minutes and mounted with mounting medium (PermaFlour by Thermo Scientific, ThermoFisher). Images were taken at an Axioplan 2 fluorescence microscope at 20x magnification using an AxioCam camera and Axiovision LE software Rel. 4.9 (all Axio devices were from Zeiss, Oberkochen, Germany). Stitching as well as computational cell detection and counting were performed in QuPath-0.2.3 (University of Edinburgh). Single cells were identified by DAPI signal and B cells, CD4 T cells and SMARTA CD4 T cells by their respective fluorescence coupled antibody (FITC/B220, APC/CD4 and PE/CD90.1). B cell zones were marked manually, and cell numbers counted computationally.

The corneal tissues were fixed with 4% paraformaldehyde at 4 °C for 30 min. Non-specific absorption was blocked in the samples using a 5% solution of normal donkey serum in Tris-buffered saline and permeabilized with 0.3% Triton X-100. The tissues were next incubated at 4 °C, for 2 days, with primary antibodies against the POU class 6 homeobox 2 (POU6F2) protein (1:100; RRID: AB_11149941; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and ZO-1 (1:100; Cat #13663; Cell Signaling Technology, Danvers, MA, USA) in Tris-buffered saline, containing 1% normal donkey serum and 0.3% Triton-X 100. The tissues were incubated with Alexa Fluor-568-conjugated anti-mouse IgG and Alexa Flour-647-conjugated anti-rabbit IgG (RRID: AB_2534013 and RRID: AB_2536183; Life Technologies) at room temperature for 2 h. They were then counterstained with 5 μg/mL Hoechst 33342 (Life Technologies). For secondary staining, the tissues were incubated with Alexa-488-conjugated antibodies against p75 neurotrophin receptor (p75NTR) (1:100; RRID: AB_10972736; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) at 4 °C overnight and mounted with PermaFlour (Thermo Fisher Scientific). The specimens were observed under a confocal fluorescence microscope (LSM710; Carl Zeiss, Jena, Germany).

The induced neurons at PID 45 were washed with PBS, fixed with 4 % paraformaldehyde (PFA) solution (Wako 163-20145) for 15 min at room temperature, and washed 3 times with PBS. The cells were simultaneously permeabilized and blocked with a solution of 5 % fetal bovine serum (FBS) and 0.3 % Triton X-100 in PBS and incubated with the primary antibodies overnight at 4 °C. On the next day, the cells were washed 3 times with PBS and incubated with secondary antibodies for 1 h at room temperature. After cell nuclei were stained with Hoechst 33258 (Dojindo Laboratories) diluted in PBS for 15 min, the cells were washed twice with PBS, 1 time with MilliQ water, and mounted with the PermaFlour (Thermo Fisher Scientific TA-030-FM). Images of the cells were captured using a fluorescence microscope (BZ-X810; Keyence, Osaka, Japan) and a confocal microscope (Zeiss LSM700; ZEISS Group, Oberkochen, Germany). Primary and secondary antibodies are summarized in Supplementary Table 2 with the dilution ratios.