Vectashield antifade reagent

Three mice were perfusion-fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS). Then, the skull was post-fixed for 10 h, decalcified in PBS containing 250 mM EDTA for 7 days, cryo-protected by overnight incubation in PBS containing 30% w/v sucrose, mounted in OCT Embedding Compound (Sakura Finetek, Tokyo, Japan) and frontally sectioned at 25-μm thickness using a cryostat (CM3050S, Leica Microsystems, Wetzlar, Germany). Sections were then incubated at room temperature overnight in an appropriate blocking solution containing an anti-CAPN5 primary antibody (ab28280, Abcam, CA, USA), washed with PBS containing 0.3% Triton X-100 (PBS-X), incubated with DAPI and the appropriate secondary antibody (Alexa-Fluor 594 conjugated, 1:200; Molecular Probes, USA; in blocking solution) for 1.5–2 h, washed in PBS-X, mounted onto MAS-coated glass slides (Matsunami Glass, Osaka, JAPAN), coverslipped using Vectashield antifade reagent (Vector Labs, CA, USA) and tightly sealed. The fluorescence signals were detected using a fluorescent microscope (BX50; Olympus, Tokyo Japan) and were analyzed with cellSens image analysis software (Olympus, Tokyo, Japan). The images for CAPN5-IR were captured by exposing the samples for 40 ms at low magnification (× 10, objective) and for 80 ms at high magnification (× 60) and were analyzed offline with NIH ImageJ (MD, USA).

In a subset of cells 0.2% biocytin was included in the intracellular solution to allow post-hoc morphological identification of GoCs. Slices were post fixed in 4% paraformaldehyde (PFA) for 24 hr. Free floating sections were washed with 1X PBS and rinsed with 0.3M glycine and 0.5% Triton-X 100. Slices were blocked in TBS containing 10% normal goat serum, 3% bovine serum albumin, 1% glycine, and 0.4% Triton-X 100 for 1 hr at room temperature. After the initial block, slices were incubated with Streptavidin conjugated Alexa 647 (1:1000; Invitrogen, Waltham, MA) overnight at 4°C. In cases of transgenic CRH-ires-CRE floxed ChR2-EYFP, slices were also incubated (overnight at 4°C) with rabbit anti-EGFP (1:1000; Invitrogen) to amplify staining of the EYFP-fused ChR2 protein. After staining, slices were rinsed with PBS and mounted with Vectashield anti-fade reagent (Vectorlabs, Burlingame, CA). Images of cells (Alexa 647) or CFs (ChR2-EYFP) were acquired using a 20X oil-immersion objective (0.85 NA) on an Olympus FluoView 300 confocal microscope using a 633 nm or 488 nm excitation wavelength. Images were processed using ImageJ software (NIH, Bethesda, MD).

To analyze intracellular protein localization by immunofluorescence, cells grown on glass coverslips were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 for 15 min, blocked with 1% goat serum–PBS, incubated with anti-SARS nsp3 polyclonal antibody in blocking buffer, and subsequently labeled with secondary antibody. Labeled cells were counterstained with TO-PRO-3 (Fisher Scientific) to visualize the nuclei and then mounted on slides with Vectashield antifade reagent (Vector Laboratories, Burlingame, CA). Images were captured using a Leica STED SP8 confocal laser scanning microscope, and images were analyzed using LAS X software.

After the treatments, all samples were fixed in 3.7% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 for 7 min and stained with rhodamine conjugated to phalloidin (Rh-Ph, 2 μg/mL, Invitrogen, USA) for 30 min at 37° C. The nuclei counterstaining was performed with Hoechst 33342 (2 μg/mL, Sigma, USA) for 10 min. After each step, the samples were washed three times with PBS. Before the imaging, the samples were mounted on glass slides using Vectashield antifade reagent (Vector Labs, USA). Samples were visualized on Olympus FV 3000 laser scanning confocal microscope (Olympus Corporation, Japan) with a 40×/1.3NA oil objective. Lasers emitting at 561 nm or 460 nm and appropriate detectors for Rh-Ph or Hoechst 33342 were used to visualize F-actin or cell nuclei, respectively. The imaging parameters (laser power, detector gains and offsets) were kept constant between all the samples. To imitate the widefield fluorescence microscope we fully opened the pinhole (airy disk) of the laser scanning confocal microscope.

Detection of the expression and localization of proteins of interest by indirect immunofluorescence assay (IFA) was performed by fluorescence microscopy as previously described [81 (link)–82 (link)]. P. yoelii wildtype or epitope-tagged sporozoites and liver stage parasites were collected at the indicated time points and were stained with primary antibodies specific to PyCSP (mAb Clone 2F6 [83 (link)]), PyTRAP (mAb Clone F3B5), PyUIS4 (rabbit polyclonal antisera [84 (link)]), HA epitope tag (rabbit polyclonal antisera, SCBT Cat#Y-11 or mAb Clone 12CA5, Roche Cat#11583816001). Alexa Fluor 488-labeled secondary antibodies against mouse or rabbit IgG were used to detect the proteins of interest, and then sporozoites were stained with 4’,6’-diamidino-2-phenylindole (DAPI) to visualize nucleic acid. Slides were mounted with VectaShield antifade reagent (Vector Laboratory) and images were acquired at 100× using an Olympus IX70 DeltaVision microscope using the softWoRx software package.

Organoids and marmoset’s corneas were fixed in 4% paraformaldehyde for 4 h at 4 °C followed by washing in PBS three times for 15 min. Organoids and marmoset’s corneas were allowed to sink in 30% sucrose overnight and then embedded in OCT and cryosectioned at 12 µm. Marmoset’s corneas were vertical embedded and sectioned. Sections were permeabilized in 0.2% Triton X-100 in PBS and blocked in block buffer (2% BSA 5% fetal bovine serum) for 1 h at room temperature. Sections were subsequently incubated with the indicated primary antibodies at a 1:100 dilution in block buffer at 4 °C overnight. Secondary antibodies used were donkey Alexa Fluor 488, 568 and 647 conjugates (Invitrogen, 1:1000). After staining with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) in PBS for 5 min, slides were mounted in Vectashield anti-fade reagent (Vector Laboratories). Confocal imaging was performed with Leica TCS SP8 DLS LightSheet microscope. Primary antibodies: PAX6 (rabbit, ab5790), CHX10 (rabbit, ab133636), ZO-1 (mouse, ThermoFisher ZO1-1A12), MAP2 (chicken, ab5392), S100β (rabbit, ab52642), RAX (rabbit, ab23340), CD31 (mouse, ab23340), aSMA (rabbit, ab5694), DAPI (49,6-diamidino-2-phenylindole), ThermoFisher D1306, NeuN (mouse, Sigma-Aldrich MAB377), AAV (rabbit ab45482), Cas9 (Sp)(E7M1H) XP^® (rabbit, CST #19526, CK3 (mouse, ab68260).