Alexa fluor 594 conjugated anti mouse igg h l

To improve detection sensitivity, the Scg3-Phage or VEGF-Phage was amplified in BLT7FLAG bacteria to label each phage particle with ~400 copies of FLAG tag [46 (link)]. Amplified phages were purified for in vivo ligand binding as above, followed by sequential intracardial perfusions with 40 mL PBS for 4 min, 20 mL of 4% PFA for 2 min and then 10 mL PBS for 1 min. Retinas were isolated, permeabilized, blocked in Solution A (5% goat serum and 1% Triton X-100 in PBS) overnight at 4 °C, and incubated with anti-FLAG M2 mAb (Sigma, St. Louis, MO, USA; #F1804; 1:200) and rabbit anti-CD31 pAb (Abcam, #28364; 1:50) in Solution A for 2 days at 4 °C. After washing, retinas were incubated with Alexa Fluor 594-conjugated anti-mouse IgG H&L (Cell Signaling, Danvers, MA, USA; #8890S; 1:1000) and Alexa Fluor 488-conjugated ani-rabbit IgG (H + L) (Cell Signaling, #4412S, 1:1000) in Solution A overnight at 4 °C. Retinas were washed, flat-mounted in 50% glycerol in PBS and viewed under a Keyence structured illumination fluorescence microscope (SIM; Osaka, Japan; Model BZ-X800).

Frozen sections of lacrimal gland tissue were fixed with methanol/acetone (1:1), blocked using 5% normal goat serum (WAKO)/0.3% Triton×-100 (Sigma) in phosphate-buffered saline (PBS), and stained with FITC anti-mouse CD326 (EpCAM) (118207, BioLegend), AdipoR2 (sc-514045), and PPARγ (2443) antibodies. Alexa Fluor594-conjugated anti-mouse IgG (H+L) (8890, Cell Signaling Technology) and Alexa Fluor 555-conjugated anti-Rabbit IgG (H+L) (A21428, Thermo Fisher Scientific) were used as secondary antibodies. These antibodies were diluted with Can Get Signal immunostain solution (Toyobo). After washing 3 times with PBS, nuclear DNA was stained with ProLong Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific). Sections were observed using a fluorescence microscope (KEYENCE) at a magnification of 400× or 1000×.