Anti mouse alexa fluor 488

HEK293T cells were grown and transfected on poly-D-lysine-coated coverslips. Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min at room temperature. They were then washed with PBS 3 times for 5 min each and permeabilized with 0.1% Triton X-100 in PBS for 15 min. Following three five-minute washes in PBS, cells were blocked with 5% bovine serum albumin (BSA) for 45 min. Incubation with primary antibodies (RP3-CPO from Triple Point Biologics, 1:1000 dilution; mouse HA-tag (6E2) antibodies from Cell signaling Technology, 1:1000 dilution) diluted in 5% BSA in PBS was performed for one hour at room temperature. After three five-minute washes in PBS, secondary antibodies (anti-rabbit Alexafluor-555 and anti-mouse Alexafluor-488 from Cell signaling technology, 1:1000 dilution) were added and incubated for one hour. Following three five-minute washes in PBS, coverslips were inverted on 8 μl of aqueous mounting medium (PBS containing 1 mg/ml p-phenylenediamine and 1 μg/ml DAPI) and sealed with nail polish. Imaging was performed on a Keyence BZ-X810 fluorescence microscope.

After A549 epithelial cells were cultured on glass coverslips (12 mm) for 72 h and infected with yeasts of P. brasiliensis, cells were washed three times with PBS, fixed with paraformaldehyde 4% for 20 min, incubated with 50 mM ammonia chloride for 20 min and with PGN/saponin (PBS containing 0.25% gelatin, 0.1% saponin, and 0.1% azide) for 1 h. Subsequently, the coverslips were incubated with the primary antibodies anti-α3 integrin, anti-Rab5 or anti-LAMP-1, diluted 1:50 in PGN/saponin for 2 h at room temperature. The coverslips were then incubated with secondary antibodies conjugated to fluorophores: anti-mouse Alexafluor 488 (#4408S Cell Signaling Technology, Danvers, MA, USA) or Alexafluor 555 anti-rabbit (#4413S Cell Signaling Technology, Danvers, MA, USA). DAPI (4′,6-diamidine-2-phenylindole) (Sigma–Aldrich/Merck, Burlington, MA, USA) was used for nuclear staining and Calcofluor (Sigma–Aldrich/Merck, Burlington, MA, USA) for the staining of the P. brasiliensis wall. The coverslips were mounted on glass slides with SlowFade^® (S36937, Invitrogen, Waltham, MA USA), and the fluorescence was analyzed with an epifluorescence microscope (Olympus BX51, Tokyo, Japan). Images were taken with the Olympus DP71 camera. Colocalization of α3 integrin and Rab5 or LAMP-1 was analyzed using Image J software (National Institutes of Health, Bethesda, MD, USA).

Primary antibodies, Anti‐GAD1 (#41 318), Anti‐NeuN (#94 403), Anti‐β‐Actin (#3700), Anti‐PSD95 (#36 233), Anti‐Caspr2 (#3731), Anti‐nNOS (#4231), Anti‐synaptophysin (#36 406) and secondary antibodies, Anti‐Rabbit Alexa fluor 594 (#8889), Anti‐mouse (Alexa Fluor 488 (#4408), HRP‐conjugated anti‐rabbit (7076S), HRP‐conjugated anti‐mouse (7074S), ProLong gold Antifade with DAPI (#8961), and protease phosphatase inhibitor cocktail (#5872) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti‐3Ntyr antibody (ab110282), Anti‐homer 1 (ab184955), Anti‐NMDAR1 antibody (ab109182), Anti‐SLC32A1/VGAT antibody (1b235952), and anti‐MAP2 (ab11268) were purchased from Abcam, Cambridge, UK. Anti‐Shank3 (sc‐377088) was purchased from Santa Cruz Biotechnology, Dallas, TX, USA. Other general chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA) and Bio‐Rad Laboratories (Haifa, Israel).