Donkey anti goat igg h l alexa fluor 488

Eyes (n = 6/group) were collected on day 1 after OAB and fixed in 4% paraformaldehyde at 4 °C overnight. Then, 10% sucrose was used to dehydrate for 1 day and replaced with 20% sucrose for 8 h, followed by 30% sucrose overnight. Afterward, the eyeballs were embedded in optimal cutting temperature compound (Sakura Finetechnical Co., Tokyo, Japan) at −80 °C and sectioned into 10 μm slices through the optic disc. TUNEL staining (in situ Cell Death Detection Kit, TMR red, version 12, Roche, Basel, Switzerland) was performed according to the manufacturer’s instructions. For double staining of TUNEL and Brn3a, sections were first stained with goat polyclonal antibodies against Brn3a (1:500; Santa Cruz Biotechnology, Dallas, TX, USA) at 4 °C overnight, and then incubated with donkey anti-goat IgG H&L (Alexa Fluor 488; 1:500; Abcam, Cambridge, UK) secondary antibody together with TUNEL staining. The retinal sections were then stained with 4,6-diamidino-2-phenylindole (DAPI) and photographed using a confocal microscope (Zeiss LSM800 or Zeiss LSM880).

For the analysis of the HA expression in the transfected HEK293 cells, they were fixed in 4% PFA (paraformaldehyde) for 30 min at 40°C, washed with PBS and permeabilized in 0.1% Triton X-100. The cells were then incubated with primary goat Anti-Influenza A Antibody (Chemicon^®, Sigma-Aldrich, #AB1074) in PBS with 0.1%/0.02% BSA/Triton X-100 at 40°C overnight. The next day, cells were incubated with Donkey Anti-Goat IgG H&L (Alexa Fluor^® 488) (ab150133; Abcam) secondary antibodies for 1 h at room temperature. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (300 nM). Images were acquired on ZOE Fluorescent Cell Imager (Bio-Rad).

Immunofluorescence staining was performed on fixed RANKL‐primed BMDMs, blocked with 5% BSA and incubated overnight with anti‐Galectin‑3 mAb (R&D Systems, Cat# AF1197) and anti‐LAMP‐1 (CD107a) Antibody (Sigma Aldrich, Cat# AB2971). The cells were, then, incubated with Alexa Fluor^® 488 Goat anti‐Rat IgM Antibody (Biolegend, Cat# 408908), Alexa Fluor^® 488 Donkey Anti‐Goat IgG H&L (Abcam, Cat# ab150129) and Alexa Fluor^® 405 Goat Anti‐Rabbit IgG H&L (Abcam, Cat# ab175652). The nuclei were counterstained using Draq5^TM (Thermo Scientific, Cat#62254) staining solution, and the slides were mounted using Aqua‐Poly/Mount (Polysciences, Cat# 1860620). The images were acquired using a Zeiss LSM 800 confocal microscope at 20 and 40× magnification and analyzed using the Zen (Black edition), Zen 3.2 (Blue edition) and Image J software.

For cryoprotection, brains were gradually immersed in 10, 20, and 30% sucrose in 24 h intervals and embedded in OCT compound (Leica Microsystems, Bensheim, Germany) with liquid nitrogen. The brains were cut into frozen coronal sections (30 μm) using a Leica CM3050 cryostat and then stored in a free-floating buffer. For immunohistological analysis, the sections were blocked in 5% normal chicken serum (containing 0.3% Triton X-100) for 1 h. After washing, the sections were incubated with primary antibodies against ionized calcium-binding adaptor molecule 1 (Iba-1, 1:200, 019-19741, Wako) or 5-hydroxytryptamine (5-HT, 1:200, ab66047, Abcam) overnight at 4°C. Subsequently, the sections were incubated with goat anti-rabbit IgG HRP (1:400, ab6722, Abcam) or Alexa Fluor^® 488 donkey anti-goat IgG H&L (1:400, ab150129, Abcam) secondary antibodies for 2 h at RT. To analyze the Iba-1-positive cells, the sections were incubated with an avidin-biotin-peroxidase complex (VECTASTAIN ABC kit, Vector Laboratories) for 2 h. The peroxidase activity was visualized using a stable diaminobenzidine solution. To analyze the 5-HT-positive signal, the sections were exposed to DAPI (1:1,000, D9542, Sigma) to stain cell nuclei. Immunoreactivity was observed under an Axio-Phot microscope (Carl Zeiss, Germany). The intensity was quantified using ImageJ 1.46 software (NIH, Bethesda, MD, USA).

LepR expression on naïve CD4⁺ T cells and T_FH cells from WT mice were stained with leptin receptor (LepR) (Polyclone, R&D) with secondary antibody Alexa Fluor 488 Donkey Anti-Goat IgG H&L(Abcam).
Leptin-secreting cells in the spleen of WT mice were stained with the following antibodies conjugated with fluorochromes: Alexa Fluor 488 anti-mouse IgM (Clone: RMM-1, Biolegend), Brilliant Violet 421 anti-mouse CD4 (Clone: GK1.5, Biolegend), Alexa Fluor 555 leptin (Polyclone, Bioss), Brilliant Violet 421 anti-mouse CD19 (Clone: 6D5, Biolegend), Alexa Fluor 488 anti-mouse F4/80 (Clone: BM8, Biolegend), Nuclei were identified by DAPI staining. Images were obtained with confocal microscopy (Zeiss LSM 710, Zeiss), and analysed with Photoshop CS4 software (Adobe) and Zen software (Zeiss).