Axioskop 2 mot microscope

Selected brains were isolated, processed, embedded, sectioned, and stained by the Histopathology Facility at the Vienna Biocenter Core Facilities (VBCF), member of the Vienna Biocenter (VBC), Austria. Briefly, 2-μm-thick coronal or sagittal paraffin-embedded sections were prepared by routine microtomy and stained with hematoxylin and eosin (H&E, Shandon* Harris Hematoxylin Acidified; Thermo Scientific Shandon Eosin Y; Fisher Scientific), Luxol Fast Blue–Cresyl Violet (LFBCV, Sigma), or with anti–Myelin Basic Protein antibody (Abcam, 1:100). Slides were imaged using a Zeiss Axioskop 2 MOT microscope (Carl Zeiss Microscopy) and subsequently digitized with the Pannoramic FLASH 250 II automated slide scanner (3D Histech). Images were acquired with the Pannoramic Viewer software (3D Histech) and with SPOT Insight camera (Diagnostic Instruments, Inc.).

Frozen sections of the draining lymph nodes were air dried for 30 min. and fixed in ice-cold acetone for 5 min. Tissue was blocked for 1 h with M.O.M.^™ Ig Blocking Reagent (Vector) diluted in PBS, washed in PBS, incubated for 5 min in the M.O.M.^™ Diluent (Vector) before incubating with mouse anti-mouse CD45.1 (Clone A20 Biolegend; 1:250 dilution in MOM diluent) for 1 h at room temperature. Slides were washed, incubated in goat anti-mouse Alexa Fluor 488 (Invitrogen, 1:300 in Dako antibody diluent) for 30 min., washed and mounted with Vetashield medium (plus DAPI for nuclear staining; Vector). Images were captured using a Zeiss Axioskop 2mot + microscope (Zeiss).

Tissues were dissected from adult mice that were anesthetized with ketamine/xylazine and PBS-perfused via the aorta. After overnight fixation at 4 °C with 4% paraformaldehyde in PBS, tissues were washed with PBS, and cryoprotected in 20% sucrose. Tissues were then infiltrated overnight at 4 °C and embedded the next day with the same 2:1 mixture of 20% sucrose and O.C.T. compound before being frozen on dry ice.
For identification of vascular cell type, tissue sections were subjected to immunoperoxidase staining. After washing with PBS and permeabilization with PBS containing 0.2% Tween 20 (PBST), endogenous peroxidase activity was blocked by incubation with 1% H₂O₂ in PBS for 10 min. Non-specific binding was blocked by incubation with blocking buffer (3% rabbit serum in PBST) for 30 min at room temperature. Tissue sections were incubated with either rat anti-mouse CD31 (1:250, BD Pharmingen) or rat anti-mouse EMCN (1:1,000, mAb V.7C7, Santa Cruz) diluted in blocking buffer overnight at 4 °C. A section incubated with rat IgG as a negative control was included in each experiment. Slides were examined and images were captured using a Zeiss Axioskop 2 MOT microscope with a mounted AxioCam digital camera under both × 20 and × 40 objective lenses. Post-acquisition processing of images was conducted using Adobe Photoshop.

Histological sections were examined under an Axioskop 2 MOT microscope (Carl Zeiss, Göttingen, Germany). Micrographs were captured with an Axiocam HR digital camera (Carl Zeiss) and the KS400 image analysis software (v3.0, Carl Zeiss). Following shading correction in KS400, images were cropped, resized, and adjusted for brightness (including slight gamma changes), colour balance, and sharpness in Corel Photo-Paint X4. All adjustments were applied to the whole image and no specific features within the photographs were modified, removed, or inserted.