Alexa fluor 594 anti rabbit

Mice were anesthetized with 5% chloral-hydrate and perfused with 4% paraformaldehyde in PBS. Brains were removed and infiltrated with 30% sucrose for dehydration. Then brains were coated with OCT compound and sliced (40 μm). Slices were blocked in 5% normal goat serum in PBS for 1 h and incubated with primary antibodies against rabbit anti-Iba1 (Wako, 019-19741, 1:1000) or rabbit anti-CD86 (Abcam, ab53004, 1:200) overnight at 4 °C. After washing in PBS for 4 times, slices were incubated with anti-rabbit Alexa Fluor 488 or anti-rabbit Alexa Fluor 594 (Invitrogen) for 1 h at room temperature. Images were photographed using fluorescent microscopy (Nikon 80i) and Imaging computer program (NIS-Elements BR, Nikon). Finally, photographs were analyzed using the NIH Image J software.

ARPE-19 cells were cultured in four-well chamber slides (Nunc Lab-Tek) to confluence and allowed to form mature epithelial structures by growing in low-serum media (5% FBS) for 3 days. The confluent monolayer was infected with C. albicans at an MOI of 1 for different time points. The cells were fixed using 4% paraformaldehyde in 1× PBS overnight at 4°C. The cells were washed thrice with 1× PBS and then blocked using 1% BSA in 1× PBS for 1 h at room temperature. The cells were then incubated with rabbit anti-ZO1 IgG (1:100) in the dilution buffer in a humidified chamber overnight at 4°C followed by three washes with 1× PBS. The cells were then incubated with anti-rabbit Alexa Fluor 594 (Invitrogen) for 1 h at room temperature in a humidified chamber. The cells were washed thrice with 1× PBS and mounted in Vectashield antifade mounting medium (Vector Laboratories). The cells were visualized using the Keyence fluorescence microscope at ×600 magnification. The cells with intact peripheral ZO-1 staining were counted against the DAPI (4′,6-diamidino-2-phenylindole)-stained cells to detect the nuclei using ImageJ software and represented as cells with ZO-1 staining/DAPI.

Serial sectioned unstained slides were incubated at 37°C for 30 minutes. Paraffin was removed though incubation in Histo-Clear (National Diagnostics, Atlanta, GA) followed by rehydration washes in 100%, 95%, 70%, 50% ethanol and deionized water. Peroxidases were neutralized with 0.3% H₂O₂ (Fisher Scientific, Fair Lawn, NJ). Slides in 10 mM citric acid (pH 6.0) were heated in a microwave for two 5 minute intervals then cooled for 20 minutes at room temperature. Slides were washed with PBS, blocked with 0.5% NEN (Boston, MA) at room temperature for 1 hour, and then incubated in anti-amylase primary antibody (1∶1000) overnight at 4°C (Sigma-Aldrich). Slides were washed and incubated in secondary antibody Anti-Rabbit AlexaFluor594 (1∶500) (Invitrogen) at room temperature for 1 hour, counterstained with DAPI, then mounted with 50% glycerol in 10 mM Tris-HCl (pH 8.0). Images were obtained using a Leica DM5500 Microscope System and captured with a Spot Pursuit 4 Megapixel CCD camera (Diagnostic Instruments, Inc., Sterling Heights, MI). Analysis was performed with ImagePro 6.3 software. Amylase-positive area was measured from twenty fields of view (FOV = 0.39 mm²) per mouse with 4 mice per treatment. Data are shown as the percent of amylase positive area to the total area of the parotid gland.

The mitotically synchronized cells were plated onto fibronectin coated coverslips (NEUVITRO, #GG-12–1.5-Fibronectin), released for 30 min, and subjected to immunocytochemistry as described previously, with minor modifications (25 (link)). The following antibodies were used: Rabbit anti-mCherry (1:500 dilution) (Abcam, #ab167453); Mouse anti-Aurora B (AIM1) (1:250 dilution) (BD Biosciences, #611082); anti-Mouse Alexa Fluor 488 (1:500 dilution) (Invitrogen, #M11032); and anti-Rabbit Alexa Fluor 594 (1:500 dilution) (Invitrogen, #M32731) (25 (link)). All cells were counterstained with DAPI/VECTASHIELD mounting media (VECTOR laboratories, #H-1200). Cell images were documented at 1000× magnification with a Nikon Ti Eclipse microscope using MetaMorph imaging software. The Z-section images of Figure 2 and Figure 3 were also documented at 1000× magnification using OptiGrid Structured Illumination microscopy (Nikon). The Z-section images of Figure 6 and Figure 7 were documented at 1000× magnification using Yokogawa CSU10 Spinning Disc Structured Illumination microscopy (Olympus).

In order to colocalize microglial phagocytic cups and fragmented nuclei, Iba1 and DAPI were used, respectively. Free-floating tissue sections from P17 cerebellums were rinsed with 0.1 m PBS, incubated with 3% hydrogen peroxide in PBS for 30 min, rinsed, and then incubated with 0.3 m glycine in 0.4% PBS-T for 60 min. Subsequently, sections were incubated with Iba1 (1:1000, Wako Chemicals) in 0.4% PBS-T containing 10% BSA for 30 min at RT with constant agitation, and then kept for 24 h at 4°C with constant agitation. After primary incubation, sections were rinsed in PBS and incubated with the secondary antibody anti-rabbit AlexaFluor 594 (1:500; Invitrogen) in PBS-T for 120 min in the dark. Sections were then rinsed, mounted and cover-slipped using Hardset mounting medium containing DAPI (Vector Laboratories).
To colocalize dead or dying cells with microglial phagocytic cups, we followed the fluorescence protocol described above to identify the cellular death marker cleaved caspase-3 (1:750, Cell Signaling Technology) and Iba1 (1:1000, Abcam) on P17 cerebellar sections (both cleaved caspase-3 and Iba1 antibodies were incubated together). Anti-rabbit AlexaFluor 488 (1:500; Invitrogen) and anti-goat AlexaFluor 594 (1:500, Invitrogen) were used as secondary antibodies.