Alexa fluor 488 conjugated anti rabbit igg secondary antibody

The tumour tissues excised from the mice treated with thrice-daily intravenous injections of the samples, as described in the western blot analysis section, were perfused with 4% paraformaldehyde (PFA), fixed overnight in 4% PFA, and cryoprotected using consecutive solutions of 10, 15, and 20% sucrose per PBS. After freezing in O.C.T. compound, 10-µm-thick sections were prepared. The sections were stained with an anti-GFP rabbit monoclonal antibody (A-6455, Invitrogen) and an Alexa Fluor^® 488-conjugated anti-rabbit IgG secondary antibody (R37116, Invitrogen) at a dilution of 1:300 for both antibodies. The TUNEL assay was performed using an in situ cell death detection kit (Roche Applied Science, Mannheim, Germany). The sections were mounted using ProLong^® Gold Antifade Mountant with DAPI (Thermo Fisher Scientific Inc.). Quantification was performed by separately counting TUNEL-positive cells (red) and total cells (blue) using ImageJ software.

The experimental protocols for the detection of NMU included minor modifications of methods described previously [13 (link), 22 (link)]. To detect NMU, mouse ears were immersed in 4% paraformaldehyde overnight, 6-mm thick sections were prepared, and the sections were reacted with a rabbit anti-NMU serum (gift from M. Kojima, diluted to 1:1000) overnight at 4°C and then with Alexa Fluor 488-conjugated anti-rabbit IgG secondary antibody (diluted to 1:200) (A-11034; Invitrogen, Carlsbad, USA). Sections incubated with an isotype-control rabbit serum instead of the primary mAb served as a negative control. Hematoxylin-eosin and toluidine blue staining were performed as described previously [17 (link)–19 (link)]. The number of mast cells in ear-tissue sections stained with toluidine blue was determined using an Olympus microscope (40× objective, 10× eyepiece) by two independent observers who were uninformed of the identities of the samples. The assessment of each observer did not differ significantly.

For anti-T4 immunofluorescence, 5-day-old larvae were fixed in 4% PFA overnight at 4°C. They were then washed in PBS containing 0.1% Tween 20 (PBST), treated with 50 µg/ml proteinase K (Roche) for 35 min and post-fixed in 4% PFA for 20 min. After several washes in PBST, larvae were incubated in blocking buffer (PBST containing 1.0% DMSO, 5% goat serum, and 0.8% Triton X-100) for 2 hr at RT, followed by overnight incubation in with anti-T4 antibody (1:2000, MP Biochemical, Solon, OH). After three washes in PBST containing 1% BSA, larvae were incubated with Alexa Fluor 488-conjugated anti-rabbit IgG secondary antibody for 3 hr at RT (1:500; Invitrogen, Carlsbad, CA). Stained larvae were washed in PBST and embedded in 1% low melt agarose for confocal imaging. For anti-E-Cadherin staining 5-day-old larvae were frozen and embedded in OCT (Sakura Finetek, Torrance, CA). Larvae were sectioned at 10 μM on a cryotome and sections were stained with anti-E-Cadherin (1:500, Abcam, Cambridge, MA), followed by anti-mouse Alexa Fluor 488.

BMDMs were cultured on Poly-D-Lysine-coated (GIBCO, Grand Island, NY, USA) cover glasses in 24-well plates at a density of 2 × 10⁵ cells/well, fixed in 3.7% formaldehyde solution (Sigma-Aldrich) for 15 min, washed with PBS and permeabilized with 0.05% Triton-X-100 (Sigma-Aldrich) for 10 min, then blocked with CAS-Block Histochemical Reagent (Invitrogen) for 30 min, all at room temperature. Subsequently, the cells were incubated with p65 primary antibody (1:500; abcam, Cambridge, UK) overnight at 4 °C, followed by incubation with an Alexa Fluor^® 488 conjugated anti-Rabbit IgG Secondary Antibody (Invitrogen) in dark at room temperature for 1 h. Cells were then incubated in the dark with DAPI (1:1000; Sigma-Aldrich) and Texas Red™-X Phalloidin (Invitrogen) for 10 min. After washing with PBS, cover glasses were mounted with VECTASHIELD Vibrance Antifade Mounting Media (VECTOR, Burlingame, CA, USA). Images were recorded using a Leica confocal microscope (Leica Microsystems, Germany) and the Leica Application Suite Advanced Fluorescence software.

RAW264.7 cells were grown on glass cover slips. For detection of LPS/MD‐2 colocalization, cells were pretreated with 10 μmol/L 25HC for 2 hours, and then incubated with Alexa Fluor 568‐conjugated LPS (6 μg/mL) (Invitrogen) for 15 minutes. The cells were fixed with 4% paraformaldehyde and blocked with 5% BSA for 1 hour. They were then incubated with anti‐MD‐2 antibody in blocking buffer overnight at 4°C, reacted with Alexa Fluor 488‐conjugated anti‐rabbit IgG secondary antibody (Invitrogen) and mounted with antifluorescence quenching medium. For nuclear translocation of NF‐κB, the cells were pretreated with or without 25HC (10 μmol/L) for 2 hours before 100 ng/mL LPS stimulation. After 1 hour of LPS treatment, cells were washed three times with PBS, followed by fixation with 4% paraformaldehyde for 15 minutes. Then, they were permeabilized with 0.2% Triton X‐100 and blocked with 5% BSA. The cells were incubated with NF‐κB p65 antibody overnight at 4 °C and incubated with Alexa Fluor 594‐labelled secondary antibody (Proteintech, Wuhan, China) at room temperature for 1 hour. Afterwards, nuclei were stained with 1 mg/mL 4′, 6‐diamidino‐2‐phenylindole (DAPI) solution (Solarbio, Beijing, China) for 5 minutes. All slides were visualized and imaged under FV3000 fluorescent microscope (Olympus).