Axio imager z1 microscope

EBs were fixed for more than days 30–90 of differentiation and immunocytochemistry performed on cryostat sections as previously described 21. Sections were reacted against a panel of retinal, lens, and corneal‐specific antibodies, listed in Supporting Information Table S1. The number of investigated structures per antibody varied slightly given the higher frequency of optic structures generated with IGF‐1; however, at least five structures from each differentiation time point across each experimental group were immunostained and this analysis was repeated for all the biological triplicates. Images were obtained using a Zeiss Axio Imager.Z1 microscope with ApoTome.2 accessory equipment and AxioVision software. For TEM, tissues were fixed in glutaraldehyde and processed by the Electron Microscopy Research Service at Newcastle University. The human embryonic and fetal material was provided by the Human Developmental Biology Resource (http://hdbr.org) under ethics permission (09/H0906/21). Due to the precious nature of this material, we restricted the use of this tissue to two sections per antibody.

Mouse brains were cut in 12 µm serial sections in the coronal plane. Tissue sections were postfixed and incubated with the primary antibodies overnight at 4 °C. Primary antibody against Qki-5 isoform (1:1000 v/v, gift of Dr Karen Artzt, Univ. of Texas at Austin and Dr Monica Justice, Baylor College of Medicine) with secondary anti-rabbit AF488 conjugated antibodies (1:1000 v/v, ThermoFisher A21206, CA) were used. DAPI (4′,6-diamidino-2-phenylindole)-counterstained sections were photographed using a Carl Zeiss AxioImager Z1 microscope and AxioVision Digital Image Processing System version 4.8.2.

Cryosections were processed as for immunofluorescence, up to and including Fab fragment treatment and incubation with primary antibodies. Duolink was performed as per manufacturer’s instructions (Olink Bioscience) using anti-rabbit PLUS and anti-mouse MINUS PLA probes and Orange detection reagents. Imaging was performed using a Zeiss Axio Imager Z1 microscope with an AxioCam MRm camera attachment running AxioVision software release 4.8.

Patient-derived neutrophils for immunofluorescence analysis of LC3B conversion were incubated overnight in a humid chamber at 4 °C with primary antibody anti-LC3 (anti-rabbit, Sigma-HPA003595). The next day, cells were washed three times in PBS for 5 min and incubated with secondary antibodies at a dilution of 1:200 and PE-conjugated (anti-rabbit, Sigma-F0382) for 1 h at room temperature. The slides were mounted with medium containing DAPI (4′,6-diamidino-2-phenylindole, Santa Cruz Biotechnology, Santa Cruz, CA, USA) to visualize nuclei. Coverslips were observed using a Zeiss Axio Imager Z1 Microscope with Apotome 2 system (Zeiss, Milan, Italy), equipped with an AxioCam camera (Zeiss, Jena, Germany). After first observation of the tissue sections under a 20× objective, we identified five fields with the largest number of immunostained cells. Then, using 40× oil-immersion objective, the immune-positive cells were counted in each one of these fields. The numerical aperture was 1.35 (40× lens), and images of HDNs were deconvoluted with SoftWorx 3.5.0 (Applied Precision, Bratislava, Slovakia). Fluorescence intensity was quantified in an automated fashion with IN Cell Investigator software (GE Healthcare, Piscataway, NJ, USA).

The IHC was performed as described previously by our group with some modifications [70 (link),71 (link)]. Briefly, mice were sacrificed at 22 days. Livers and spleens were removed and fixed overnight in 4% formalin, then embedded in paraffin, and cut into 3 μm sections with a Rotary microtome (RM2235, Leica, Wetzlar, Germany). Immunohistochemistry was performed on the platform of the Leica company, BOND-MAX Immunohistochemical staining machine. The primary antibodies used for immunohistochemistry are anti-arginase-1 (GenomeMe, IHC400-100, 1:800, Richmond, BC, Canada), anti-CD45 antibody (Biocare, CM016, 1:200, Warrington, UK), anti-CDX2 antibody (Cellmarque, 235R-15, 1:200, Rocklin, CA, USA), anti-Pan-Cytokeratin antibody (Cellmarque, 313M-14, 1:600, Rocklin, CA, USA), and anti-Vimentin antibody (Novocastra, NCL-L-VIM-V9, 1:500, Deer Park, IL, USA) with overnight incubation. A labeling system (Bond Polymer Refine Detection, DS9800, Leica) containing secondary antibodies labeled with horseradish peroxidase (HRP) and DAB-3 (3′-diaminobenzidine) was used as the chromogen for antigen signal detection. Hematoxylin (ready to use, Leica) was used for contrast staining. The Zeiss Axio Imager Z1 microscope (ocular 10×, objectives 10×, 40×) was used for visualization with the Zeiss AxioCam MRm camera and AxioVision SE64 4.9.1 software (Carl Zeiss AG, Oberkochen, Germany).