Tcs sp8x white light laser confocal system

Small intestines containing PPs were harvested from adult mice, washed with PBS, fixed in 4% PFA for 1 hour or overnight and immersed in 2% agarose. 40-70μm sections were obtained using a Leica VT1200S vibratome and blocked in PBS containing 1% albumin and 0.1% saponin for 30 min. Embryo or newborn small intestine were washed, fixed in PFA 4% for 1 hour and snap-frozen in an OCT-filled mold on a liquid nitrogen-cooled metal surface. 10μm-thick cryosections were rehydrated in wash buffer (0.1% saponin in PBS) for 5 min and blocked in PBS containing 0.5% albumin for 30 min. Sections were incubated with the indicated antibodies and images were acquired with a TCS SP8X White Light Laser confocal system (Leica) and a Zeiss LSM710 confocal microscope.
For whole tissue imaging, PPs were cleared using the ScaleA2 protocol^{57 (link)} for 2 weeks or Focus Clear (CelExplorer) and samples were imaged using the ZEISS Lightsheet Z.1 or LSM710 microscope. Acquired 3D data stacks were reconstructed employing Imaris (Bitplane) software. Images were processed for noise removal and cells were identified as objects based on the intensity of fluorescence in the respective channel and filtered based on size.

Tumor samples were fixed overnight in 4% PFA/PBS, washed with PBS and a small part was immersed in 30% sucrose/PBS, embedded in OCT (VWR Chemicals) and frozen, while the rest was embedded in paraffin. 4-μm-thick sections of FFPE (Formalin-Fixed, Paraffin-Embedded) tumors were blocked using 1% BSA in TBS containing 0.05% Tween 20 (Sigma) (TTBS) and incubated with anti-Ki67 (Thermofisher Scientific, MA5-14520) or anti-CD31 (Abcam, ab28364) primary antibodies. A biotinylated secondary anti-rabbit antibody (Vector Laboratories) and the Vectastain ABC kit (Vector Laboratories) were used for signal detection and amplification, respectively. Signal development was performed with the Vectastain DAB (3,3-diamino-benzidine) kit (Vector Laboratories) and hematoxylin was used as a counterstain. Images were acquired with an Eclipse E800 microscope (Nikon) equipped with a QImaging Digital Camera. Cryosections were incubated with the anti-CD31 and anti-VCAM1 (Abcam, ab19569) antibodies, followed by the secondary A568-conjugated anti–rabbit antibody (Invitrogen, A110011) and the A488-conjugated anti-rat antibody (Invitrogen, A11006), respectively. Mounting medium containing DAPI (Sigma-Aldrich) was used to stain the nuclei and images were acquired using a Leica TCS SP8X White Light Laser confocal system. Quantifications were performed using ImageJ software analysis