Alexa fluor 594 conjugated goat anti rabbit igg

TSPCs were fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 for 20 min, and then blocked with 3% bovine serum albumin for 1 hr at room temperature. The cells were washed and then incubated with primary antibodies at 4°C overnight, followed by incubation with a mixture of Alexa Fluor 594-conjugated goat anti-rabbit IgG (Cell Signaling Technology). The nuclei were counterstained with DAPI. Immunofluorescence was visualized under a Nikon fluorescence microscope.

Briefly, cells grown on coverslips in 12-well plates were fixed with freshly prepared 4% paraformaldehyde in 0.01 mol/L PBS for 30 min. After washing with PBS, the cells were blocked with 10% normal goat serum (Solarbio) for 1 h at room temperature. Then, cells were incubated with the following primary and secondary antibodies: rabbit anti-von Willebrand factor (1:400, Abcam, Cambridge, MA, USA), rabbit anti-CD31 (1:200, Abcam), mouse anti-LMP2, mouse anti-occludin and mouse anti-claudin-1 (1:100, Santa Cruz Biotechnology, USA), rabbit anti-β-catenin (1:200, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-ZO-1 (1:100, Invitrogen), Alexa Fluor® 594 conjugated goat anti-rabbit IgG or Alexa Fluor® 488 conjugated goat anti-mouse IgG (1:1000, Cell Signaling Technology). Finally, slides were mounted in antifade mountant with DAPI antifade reagent (Invitrogen) prior to imaging. The mean immunofluorescent intensity of each target protein was calculated using the software ImageJ (Version 1.53 m) and followed the literature [18 ].

Sporozoites resuspended in PBS were dried onto microscope slides, whereas intracellular stages of C. parvum in HCT-8 cells were grown on coverslips for 24 and 48 h. These slides or coverslips were fixed with methanol at room temperature for 15 min. After three washes with PBS, the fixed sporozoites or cells were treated with 0.5% Triton-X in PBS for 15 min and blocked for nonspecific binding with 5% BSA in PBS for 1 h. After three washes with PBS, they were incubated with antibodies against INS-15 domain I (~0.6 µg/mL) or INS-15 polypeptide (~3.9 µg/mL) in 5% BSA-PBS for 1 h, followed by incubation with Alexa Fluor 594-conjugated Goat Anti-rabbit IgG (Cell Signaling Technology, Beverly, MA, US) in 5% BSA-PBS at 1:400 for another hour. After three washes, the slides or coverslips were counterstained with the nuclear stain 4′,6-diamidino-2-phenylindole (DAPI, Sigma, St Louis, MO, USA). After three more washes, the slides or coverslips were mounted with No-Fade Mounting Medium (Booster, Wuhan, China) and examined using differential interference contrast (DIC) and fluorescence microscopy on a BX53 microscope (Olympus, Tokyo, Japan).

Oocysts and free sporozoites were fixed on slides with 4% paraformaldehyde in PBS for 30 min to examine CpCDPK expression in these stages. For other developmental stages, intracellular parasites in HCT-8 cell cultures harvested 24 and 48 h after infection were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. The polyclonal antibodies against CpCDPKs (5.0 μg/mL, diluted in PBS) were used as the primary antibodies, while Alexa Fluor 594-conjugated Goat Anti-rabbit IgG (Cell Signaling Technology, Danvers, MA, USA) was used as the secondary antibody in an immunofluorescence assay. 4’,6-diamidino-2-phenylindole (DAPI, Roche, Basel, Switzerland) was used to counterstain the nuclei of the organisms [23 (link)]. After being mounted with the No-Fade Mounting Medium (Boster, Wuhan, China), the slides were covered with coverslips, sealed with nail polish, and examined using the differential interference contrast (DIC) and immunofluorescence microscopy under a BX53 microscope (Olympus, Tokyo, Japan).

Free-floating 40 µm coronal hippocampal sections were collected and blocked with 0.3% Triton X-100 and 3% goat serum in 0.01 M PBS (pH 7.3) for 30 min. The slices were incubated with primary antibody to NeuN produced in rabbit (Millipore, USA), diluted at 1:200 in 0.01 M PBS, overnight at room temperature. The sections were then washed in 0.01 M PBS and incubated with secondary antibody, Alexa Fluor 594 conjugated goat anti-rabbit IgG (Cell signaling Technology, USA), diluted in 0.01 M PBS (1:200), for 4 h at room temperature. Terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end labeling (TUNEL) staining was performed using the In Situ Cell Death Detection Kit (Roche Applied Science, Germany), as described in detail previously [8 (link)]. Briefly, after NeuN staining, the mounted coronal sections were rinsed with PBS and treated with 1% Triton-100 in PBS for 2 min on ice. The sections were rinsed in PBS and incubated for 60 min at 37°C with 50 μl of TUNEL reaction mixture. The negative control sections were incubated for 60 min at 37°C with 50 μl of parallel solution without TdT buffer and biotinylated dUTP. After washing with PBS, the slices were analyzed with fluorescence microscopy. For each rat, a total of 15 visual fields in three hippocampal sections was counted for TUNEL-stained cells.

Sporozoites were dried onto microscopy slides and intracellular stages of C. parvum in HCT-8 cell cultures were grown on coverslips for 48 h. They were fixed with methanol for 15 min. After three washes with PBS, the slides were treated with 0.5% Triton-X 100 in PBS for 15 min and blocked with 5% bovine serum albumin (BSA) in PBS for 1 h. After another three washes, the slides were incubated with polyclonal antibodies against individual INS proteins (1:400 diluted in 5% BSA-PBS) for 1 h. Alexa Fluor 594-conjugated goat anti-rabbit IgG (Cell Signaling Technology, MA, United States) diluted 1:400 was used as the secondary antibodies. After 1-h incubation and three washes, the slides were counterstained with the nuclear stain 4’6-diamidino-2-phenylindole (DAPI, Roche) at room temperature for 5 min. The slides were mounted with No-Fade Mounting Medium (Booster, Wuhan, China), sealed with nail polish, and examined using differential interference contrast (DIC) and fluorescence microscopy on a BX53 microscope (Olympus, Tokyo, Japan).

For the assessment of CpINS-4 and CpINS-6 expression in developmental stages of C. parvum, oocysts and sporozoites were suspended in PBS and dried onto poly-L-lysine-treated microscope slides (Waterborne, New Orleans, LA, USA), whereas HCT-8 cells infected with sporozoites were grown on coverslips for 24 and 48 h, fixed with methanol for 15 min, and permeabilized with 0.5% Triton X-100 for 30 min. After blocking with 5% BSA at room temperature for 1 h, oocysts, sporozoites and infected HCT-8 cells were probed overnight with the polyclonal antibodies (1:500) in 5% BSA/PBS (weight/volume), followed by staining with Alexa Fluor 594-conjugated goat-anti-rabbit IgG (Cell Signaling Technology, Danvers, MA, USA) and nuclear staining with 4′, 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). After three washes with PBS, the slides and coverslips were mounted with Antifade Mounting Medium (Booster, Wuhan, China) and examined using differential interference contrast (DIC) and fluorescence microscopy on a BX53 microscope (Olympus, Tokyo, Japan).