Alexa fluor 488 goat anti rat igg

A triple-staining procedure was implemented for BrdU/DCX/NeuN to allow analyzation through immunofluorescence. The brain sections were incubated overnight at 4°C with the following primary antibodies: rat anti-BrdU (1:200, ab6326, Abcam), mouse anti-NeuN (1:500, ab104224, Abcam) and rabbit anti-DCX (1:500, ab18723, Abcam). The sections were washed in PBS and incubated for 4 h with fluorescent secondary antibodies: Alexa Fluor 488 goat anti-rat IgG to reveal immunoreactivity of BrdU, Alexa Fluor 405 donkey anti-mouse IgG to reveal immunoreactivity of NeuN, and Alexa Fluor 594 goat anti-rabbit IgG to reveal immunoreactivity of DCX, respectively (1:400 for all three antibodies, Abcam). Every sixth coronal section from the adult dentate gyrus was used to assess BrdU+/NeuN+/DCX- and BrdU+/NeuN+/DCX+ positive cells in both hemispheres (10 sections per animal, 3 mice per group). BrdU+/NeuN+/DCX- and BrdU+/NeuN+/DCX+ positive cells were observed and counted under an Olympus BX63 microscope fitted with a ×10 eyepiece lens with a ×40 objective, and their percent distribution in the BrdU positive cells was calculated.

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).

MSCs, cultured in the 12-well plate, were washed in PBS and fixed in 4% PFA for 10 min. After fixation the cells were with 5% normal serum and 0.05% Triton X-100 (Sigma-Aldrich) in PBS for 30 min at room temperature, followed by incubation with antibodies according to the protocol provided by the manufacturer. The cells were examined using a cell imaging microplate detection system-Cytation5 (BioTek) and a laser scanning confocal microscopy (Leica Biosystems). Primary antibodies (1:300) were applied as follows: Rat monoclonal anti-Nestin (Abcam ab81462), Rabbit monoclonal anti-Alpha smooth muscle actin (Abcam ab124964) and Rabbit monoclonal anti-to Cytokeratin 14 (Abcam ab181595). 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). Nuclei were counterstained with Hoechst 33,342 (1:1000) (Invitrogen).

Fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)- and allophycocyanin (APC)-conjugated specific antibodies against mouse CD11b (clone M1/70), Ly-6G (clone 1A8), NK1.1 (clone PK136) or CD3 epsilon (clone 145-2C11), an antibody against CD31 (clone MEC 13.3), and purified rat anti-mouse CD16/CD32 (Fc Block) were purchased from BD Biosciences (CA, USA). Antibody against interleukin-1β (IL-1β) (clone 11E5) was from Santa Cruz Biotechnology (CA, USA). Alexa Fluor 488 goat anti-rat IgG and Chromeo 546 goat anti-mouse IgG were obtained from Abcam (MA, USA). All other chemicals were of the highest grade available from commercial sources. Wild-type C. perfringens Strain 13 was used in this study.

After treatment without or with κ-carrageenan (0.5 and 2 mg/mL) for 2 h, MC3T3-E1 pre-osteoblasts were fixed with 4% paraformaldehyde solution at 37 °C for 15 min, treated with 0.2% TritonX-100 (Sigma) for 15 min, and non-essentially bound substances blocked in 5% bovine serum albumin (BSA) for 30 min. Expression of phospho-paxillin (p-paxillin) was analyzed by immunofluorescence staining using rhodamine-phalloidin cytoskeleton dye (Invitrogen, Fisher Scientific, Carlsbad, CA, USA) and p-paxillin pTy31 polyclonal rabbit IgG (ab32084, Abcam, Cambridgeshire, UK). The secondary antibody used was Alexa Fluor-488 goat anti-rat IgG (Abcam). Nuclei were stained with 1 µg/mL DAPI (Sigma). After glycerol mounting, cell imaging was performed by laser scanning confocal microscopy (LSCM; Nikon, A1R/A1, Tokyo, Japan). Fluorescence microscopy was also used to visualize paxillin at 488 nm wavelength, and Image J software was used for paxillin quantification.

Flow cytometry was used to detect the expression of hACE2 and the binding of S^RBD fusion proteins with HEK293T-hACE2 cells. Briefly, HEK293T-hACE2 cells were incubated with anti-human ACE2 antibody for 2 h, HEK293T cells were used as a control. Serial dilutions of S^RBD-mFc and 2xS^RBD-mFc proteins were prepared in EP tubes, diluted fusion protein was incubated with HEK293T-hACE2 cells for 2 h, HEK293T cells were used as a control. The following secondary antibodies (1:1000) were used to detect hACE2 and mFc fusion proteins respectively, including: Alexa Fluor 488 Goat anti-Rat IgG (Abcam, ab175473) and Alexa Fluor 647 Goat anti-mouse IgG (Abcam, ab150115).