Lsm 510 meta confocal microscope

Histology fresh frozen sections were fixed in 4% PFA and stained followed by hematoxylin and eosin staining. For immunofluorescence, pancreatic tumours were fresh frozen in OCT, fixed in 4% PFA or acetone, and stained according to manufacturer's protocol. Sections were stained using the polyclonal Ki67 (Rabbit Anti-Ki67, Abcam ab15580), Trypsin 3/PRSS3 (Goat anti-Trypsin, R&D Systems AF3565) and MECA-32 (Rat anti-PLVAP, in-house hybridoma supernatant). For immunofluorescence the following detection antibody were used: Donkey anti-Rabbit 488 (Life Technologies), Donkey anti-Goat Alexa Fluor 546 (Life Technologies), Donkey anti-Rat 488 (Life Technologies) and DAPI (Life Technologies). Images were acquired using a Ziess LSM 510 meta confocal microscope.

Mice used for immunofluorescence were euthanized with a high dose of isoflurane and transcardially perfused with 4% PFA. The foot paw glabrous skin tissue was dissected removing the ligaments. The samples were then fixed with 4% PFA at 4°C for 2 Hrs. Tissues were cryo-protected in 30% sucrose in PBS (4% overnight) before freezing. Frozen glabrous skin were cut (20µm slices) on a Leica CM1950 cryostat. The tissues were then blocked with 3% BSA in PBS for 1 Hr at room temperature followed by incubation with the primary antibodies. For fluorescence immunostaining, the following primary antibodies were used: PGP 9.5 (1:500), and TLR4 (1:400). The sections were then washed with PBS thrice, incubated with Alexa-fluor 488 lebeled anti-rabbit secondary antibody (1:600) and Alexa-fluor 598 lebeled anti-mouse secondary antibody (1:600) in dark at room temperature for 2 Hrs. Subsequently, samples were washed four times with PBS, mounted using ProLong Gold antifade reagent (Thermo #P36935) and images were acquired using a Ziess LSM 510 Meta confocal microscope under identical settings.

The method has been described previously (Ning et al., 2004 (link)). Rats were sacrificed after being anesthetized, and the brains were removed. The tissue was then frozen in Tissue-Tek OCT mounting medium and 15 μm coronal sections were cut. Sections were subsequently spread on microscope slides and allowed to air dry. Air dried sections were fixed in 4% paraformaldehyde in PBS for 30 min then washed 3 times in PBS for 5 min each. After post-fixation, the sections were blocked and permeabilized in 0.1 M PBS with 0.3% TX-100 (sigma) and 5% bovine serum albumin (BSA) for 1 h. Following permeabilization, a primary anti-NeuN or anti-glycine antibody (Millipore, United States) was applied overnight at 4°C. Primary antibody was removed with three washes in PBS and secondary antibody (Alexa 594 conjugated to goat anti-mouse) was applied for 1 h at room temperature. Secondary antibody was removed with three washes in PBS and the sections were observed under an Olympus BX51 microscope or a Zeiss LSM 510 META confocal microscope.

Ca²⁺ imaging was performed following standard protocols [8 (link)]. Briefly, cells were labeled with Rhod-3 from a Calcium Imaging Kit (Thermo Fisher Scientific, R10145) for 1 h at room temperature, washed, and incubated for an additional hour, to allow de-esterification of the dye. Rhod-3-labeled cells were analyzed at 37 °C using an LSM 510 META confocal microscope (Carl Zeiss, Oberkochen, Germany). Ca²⁺ oscillation⁺ cells were counted in ten randomly selected fields per well in at least three independent experiments. To count the number of beating cells, we seeded 50,000 fibroblasts per well on 12-well plates, performed cell transductions, cultured the cells with the indicated media, and then monitored cell contraction. For accurate analyses of the cell count, we used an all-in-one fluorescence microscope as described previously (BZ-9000; Keyence, Tokyo, Japan) [8 (link)]. We first acquired images of the cells in all the areas in a well with a 20× phase contrast lens by moving the motorized stage sequentially. We next moved the field to cover all the areas in a well, and counted the number of spontaneously contracting cells in each field with the 20× phase contrast lens in at least three independent experiments. The measurements and calculations were conducted in a blinded manner.