Alexa fluor 568 conjugated anti rabbit igg

Samples were blocked in 5% bovine serum albumin in phosphate buffered saline (PBS) for 1 h, rabbit anti-CD68 (1:100; Servicebio, China), rabbit anti-CD206 (1:100; Servicebio, China) and rabbit anti-iNOS (1:100; Servicebio, China) were incubated at 4° C overnight. The secondary antibodies Alexa Fluor 568-conjugated anti-rabbit IgG (1:200; Invitrogen, UK) and Alexa Fluor 488-conjugated anti-rabbit IgG (1:200; Invitrogen, UK) were applied. The nuclear was stained by 4’, 6-diamidino-2-phenylindole (DAPI, Sigma, USA). Images were captured with a fluorescent microscope (Leica TCS SP2).

Paraffin sections (4 μm) of pulmonary tissues were rehydrated and microwaved in citric acid buffer to retrieve antigens. After incubation with 10% BSA for 1 h, the sections were incubated with primary antibodies against integrin β3 (SJ1909/NBP2-67416, Novus, USA), PKM2 (AF7244, R&D, USA), fibronectin and collagen-I (72026S, CST, USA) at a dilution of 1:100 at 4 °C overnight. After washes, sections were incubated with Alexa Fluor 568-conjugated anti-rabbit IgG (Invitrogen, Carlsbad, CA) for integrin β3, PKM2 and collagen-I, fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (Invitrogen) for fibronectin at a dilution of 1:400, at 37 ℃, for 1 h in the dark. Finally, nuclei were counterstained with 4'6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, USA). Aipathwell softwell (Servicebio, China) was used to analyze the integrated optical density of the single or double stained fluorescent proteins for the semi-quantitative analysis.

Anthers fixed in 4% paraformaldehyde were transferred to PME buffer (50 mM PIPES, 5 mM EGTA, and 5 mM MgSO₄, pH 6.9) on a MAS-coated microscope slide, cut in distilled water using a scalpel, and incubated for 30 min at 25 °C. The cleaved anthers were then blocked with 3% bovine serum albumin (BSA) in PME for 60 min. The samples were placed in primary antibody solutions (rabbit anti-AGO1b or mouse anti-AGO1d, diluted 1/500 with 3% BSA in PME), degassed five times at 0.05 MPa for 2 min, and incubated overnight at 4 °C. After washing three times for 5 min with PME, the slide was placed in secondary antibody solution (Alexa Fluor 568-conjugated anti-rabbit IgG (Invitrogen, A11036) or Alexa Fluor 488-conjugated anti-mouse IgG (Invitrogen, A11001), diluted 1/200 with 3% BSA in PME), and degassed as above. The slide was incubated in a dark chamber for 2 h at room temperature, and then incubated overnight at 4 °C. The samples were washed three times with PME buffer for 5 min, incubated for 15 min at 25 °C in 1 µg/mL DAPI (Sigma, MBD0015), and washed again with PME buffer as above^{31 (link)}. Samples were mounted in ProLong Gold antifade reagent (Invitrogen, P10144). Images were captured using an LSM 780 (Carl Zeiss).

Plasmodium vivax parasites were collected from patients with malaria in Thailand and spotted onto eight-well glass slides. The slides were fixed in ice-cold acetone for 10 min, dried, and blocked in 5% BSA in PBS-T at 37 °C for 30 min. Then the slides were incubated (dual-labelled) with rabbit anti-PvMSP1-19 (1:50 dilution), rabbit anti-PvDBP (1:50 dilution), rabbit anti-PvRAMA (1:50 dilution), and mouse anti-PvMSA180 (1:100) as primary antibodies at 37 °C for 1 h. Next, the slides were stained with Alexa Fluor 568-conjugated anti-rabbit IgG or Alexa Fluor 488-conjugated goat anti-mouse IgG as secondary antibodies (Invitrogen), and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen) at 37 °C for 30 min. The slides were mounted in ProLong Gold antifade reagent (Invitrogen) and visualized under oil immersion using a confocal laser scanning microscope (FV200Olympus, Tokyo, Japan) equipped with 20× dry and 60× oil objectives. Images were captured using the FV10-ASW 3.0 viewer software.

Before infection, the bacterial cells were incubated with 20-µM CFDA SE cell trace kit reagent (CFDA; Invitrogen, #V12883) dissolved in PBS for 1 h. Then, the bacterial suspension was centrifuged at 3,000 rpm for 15 min. Afterward; the pellets were washed with PBS, centrifuged again, and resolved in PBS to the infection experiment. HeLa cells were seeded on glass coverslips and infected with bacteria previously stained by CFDA.
The cells were fixed in 4% paraformaldehyde-PBS for 10 min at room temperature, permeabilized by 0.1% Triton X-100 solved in PBS for 7 min, and washed thrice with PBS. Next, samples were blocked with 3% BSA in PBS for 1 h, washed with PBS, and incubated with primary antibodies for LC3, p62, and LAMP2 (Abcam, #ab25631) at 1:500 dilution at 4°C overnight. Next, the samples were washed thrice with PBS and treated with secondary antibodies [Alexa Fluor^® 568-conjugated anti-mouse IgG (Invitrogen, #A11004) or Alexa Fluor^® 568-conjugated anti-rabbit IgG (Invitrogen, #A11011), 1:1,000] at 4°C for 1 h. Then, the samples were washed, and the cell nucleus was stained by 4’,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI; 500 nM, Invitrogen, #D1306) at 4°C for 5 min. Images were captured using a Keyence BZ-X700 microscope and Nikon A1R confocal laser scanning microscope.

Deparaffinized sections or fixed cells were permeabilized, blocked and incubated with acetylated α-tubulin (1:200, T6793, Sigma) overnight at 4. The slides were washed and stained with Alexa Fluor 568 conjugated anti-rabbit IgG (1:1000, Invitrogen) antibody for 1 hour at room temperature. DAPI (6-diamidino-2-phenylindole, Sigma) staining was used as the counterstain for nuclei.
The same staining procedure was used for acetylated α-tubulin (1:500, T6793, Sigma), FGFR1 (1:100, ab10646, Abcam), p-FGFR1 (1:100, ab59194, Abcam), Phospho-SMAD1 (Ser206) (1:100, PA517092, Invitrogen), and p-Smad1/5/8 (1:50, sc-12353-R, Santa Cruz) staining.

Lung tissue sections were deparaffinized in xylenes and were rehydrated by passage through serial dilutions of ethanol in distilled water. Heat-induced antigen retrieval was performed in an antigen retrieval solution (ImmuoActive; Matsunami Glass Ind., Ltd., Kishiwada, Japan) with the use of a microwave, twice, for 5 min. The sections were incubated with Blocking One Histo (Nacalai Tesque) in order to block non-specific reactions. Anti-mouse B220 (eBioscience), anti-mouse CD3 (Cell Signaling Technology) monoclonal antibodies (mAbs), and anti-mouse CD23 (Boster Biological Technology) polyclonal Ab were applied to the sections overnight, at 4°C. After washing with phosphate-buffered saline (PBS), the sections were incubated with an Alexa Fluor 488-conjugated anti-rat IgG (Invitrogen) or an Alexa Fluor 568-conjugated anti-rabbit IgG (Invitrogen) Ab. After washing thrice with PBS, the nuclear DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, Waltham, MA), and the sections were observed under an optical microscope (ZEISS Axio Observer 7; Carl Zeiss) and were analyzed through the use of ZEN3.2 blue edition (Carl Zeiss).