Alexa fluor 594 goat anti rat igg

Lung tissues were washed in PBS and fixed in 10% PBS-buffered formalin for 24 h. Tissues were then embedded in optimal cutting temperature compound, and 5–6 μm sections were cut by Microtome Cryostat (Leica CM3050S, Leica Biosystems), and stained with antibody according to the protocol provided by the manufacturer. Cells, cultured on the microscope cover glass, were washed in PBS and fixed in 10% PBS-buffered formalin for 15 min, and then stained with antibodies according to the protocol provided by the manufacturer. The sections were examined using a laser scanning confocal microscopy (Leica Biosystems). Primary antibodies (1 : 200) were applied as follows: Rat anti-mouse C3 (Abcam ab11862), Rabbit anti-C3 (Abcam ab200999), Rat anti-mouse Nestin (Abcam ab81462), Rat anti-mouse Ly6G (Abcam ab25377), Rabbit anti-human C3 (Abcam ab97462), Mouse monoclonal anti-human Nestin (Abcam ab22035), Rabbit anti-Histone H3-cit (Abcam ab5103), and Rabbit anti-Myeloperoxidase (Abcam ab208670). The following secondary antibodies (1 : 1000) were used: Alexa Fluor 488 Goat anti-Rat IgG (Abcam ab150157), Alexa Fluor 488 Goat anti-Rabbit IgG (Abcam ab150077), Alexa Fluor 555 Donkey anti-Rabbit IgG (Beyotime A0453), Alexa Fluor 647 Goat anti-mouse IgG (Beyotime A0473), and Alexa Fluor 594 Goat anti-Rat IgG (Abcam ab150160).

The mouse ears were fixed in 10% formamide and embedded in paraffin. Sections (2 μm) were deparaffinized and autoclaved at 121°C for 10 min in 10 mM sodium citrate buffer (pH 6.0). The sections were blocked with 5% skimmed milk in TBS-T at room temperature for 60 min and then incubated with rabbit anti-mMCP-6 antibody (R&D Systems, MN, USA, 1:500) at 4°C overnight. After washing with TBS, sections were incubated with secondary antibody (Alexa Fluor 594 goat anti-rat IgG, Abcam, 1:500) and FITC-avidin (BioLegend, CA, USA, 1:200) for 1 h at room temperature and counterstained with DAPI. FITC-avidin was used to label mast cells in the skin. Images were captured using a BZ-X800 fluorescence microscope (KEYENCE, Osaka, Japan). The digitized images were transferred to a computer to measure the size of each region using a software (ImageJ, National Institute of Mental Health, MD, USA), and FITC-positive mast cells were counted. The results are expressed as positive cells per mm².

Immunofluorescence staining was performed according our previous study [67 (link)]. Brain sections from cerebral cortex and cultured cells on coverslips were fixed in 4% paraformaldehyde. Samples were blocked with 5% BSA prior to incubation with primary antibody overnight with the following antibodies: the M (LPS) marker iNOS or the M (IL-4) marker Arg-1, the microglial marker F4/80 and the neuron marker NeuN (Abcam, USA). After washing with PBS, sections or slides of cells were then incubated with the appropriate secondary antibodies, Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 594 goat anti-Rat IgG (Abcam, USA), for 1 h at room temperature. After washing, the samples were restained with 4',6-diamidino-2-phenylindole (DAPI) for 12 min before mounting. Five random fields of each sample were observed and photographed with a fluorescence microscope (Olympus BX51TRF, Olympus Co., Tokyo, Japan).