Eclipse te200 u

At the indicated timepoints after KA injection, mice (WT, Atf6b^−/−, and Calr^+/−) were deeply anesthetized with isoflurane and transcardially perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde prepared in 0.1 M phosphate buffer (pH 7.4). Brains were harvested, post-fixed with 4% paraformaldehyde for 8 h, and cryoprotected in 30% sucrose for at least 24 h. Cortical Sects. (10 μm-thick coronal sections containing the hippocampus (between Bregma − 1.5 and − 2.1 mm)) were cut on a cryostat (Leica Biosystems, Wetzler, Germany). Sections were processed for Nissl staining (Cresyl violet staining) or immunohistochemistry with antibodies against cleaved caspase-3 (Asp175; Cell Signaling Technology, Inc. Danvers, MA, USA; 1:500) and c-Fos (PC05; Merck; 1:200). Nuclei were visualized with 4′,6-diamidino-2-phenylindole (DAPI; Sigma). Anti-rabbit and anti-mouse Alexa Fluor 488-conjugated (Life Technologies; 1:200) and Cy3-conjugated (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA; 1:200) secondary antibodies were used to visualize immunolabeling. Imaging was performed using a laser scanning confocal microscope (Eclipse TE200U; Nikon, Tokyo, Japan) and Nikon EZ-C1 software or using a light and fluorescence microscope (BZ-X700; KEYENCE, Osaka, Japan).

Immunostaining for Ki-67 was processed to estimate cell proliferation, in formalin-fixed paraffin-embedded tumor tissues, according to the manufacturer’s protocol. Briefly, slides were deparaffinized by heating at 55°C for 30 min, then exposed to xylene, and rehydrated via a series of descending series of ethyl alcohol solution. After that, we incubated slides in antigen retrieval solution (Dako) at 95°C for 30 min. Endogenous peroxidases activity was blocked by putting slides in methanol containing 3% hydrogen peroxide for 15 min. The slides were incubated with Ki-67 primary antibody (Abcam, Cambridge, MA) at 4°C overnight. Then, secondary antibodies were applied to the sections at room temperature for 1 h. Afterward, the sections were counterstained with hematoxylin for 30 s and analyzed by microscopy (Nikon Eclipse TE-200-U).

Experiments were performed in the “whole-cell” configuration using the internal and external solutions described below. Synaptic activity was recorded after a stable baseline was reached. Recording pipettes were pulled from borosilicate glass (WPI, Sarasota, FL) in a horizontal puller (Sutter Instruments, Novato, CA). Membrane currents were measured using an Axopatch-200B amplifier (Axon Instruments, Inc., Burlingame, CA) and an inverted microscope (Nikon, Eclipse, TE200-U, Japan). Data was collected, stored and analyzed using a data acquisition system card (Axon Instruments, Inc.) and the pClamp9 software (Axon Instruments, Inc.). For synaptic activity records, data was analyzed using the Minianalysis software, obtaining the frequency, amplitude and decay time of the records. All experiments were performed at room temperature (20–25°C) using a holding potential of −60 mV. Data are given as means ± S.E.M. and are obtained from at least 3 experiments.

To confirm identification of F-actin by phalloidin staining, retinal flat mount samples were prepared, as previously described [25 (link)]. Briefly, after deep anesthesia, mice were sacrificed, and enucleated eyes were fixed in 2% paraformaldehyde for 2 h. The RPE complex was incubated in permeabilization solution (1% Triton-X 100 and 0.3% BSA in PBS) at room temperature (RT) for 2 h. Then, the eyes were incubated at RT for 2 h with primary antibody Alexa Fluor 568 Phalloidin (1:100, Thermo-Fisher Scientific, Waltham, MA, USA). After washing with PBS, the RPE/choroid complex was mounted with Fluoromount™ Aqueous Mounting Medium (Sigma Aldrich, St. Louis, MO, USA) and observed under confocal microscope (Eclipse TE200-U; Nikon UK Ltd., 1 The Crescent, Surbiton, UK). Z-stack confocal images of RPE flat mounts were reconstructed using the NIS Element (Nikon, Shinagawa, Tokyo, Japan) software. The border of each RPE cell was outlined based on Phalloidin (F-actin) staining.

Mouse eyes were fixed in 2% PFA at room temperature for 2 h. The retinas were dissected as previously described [24 (link)]. Samples were incubated with 1% Triton X-100 in PBS for 4 h, and blocked by incubation with 1% BSA. Retinal flatmounts were incubated with rabbit anti-cone arrestin (Millipore, Watford, UK) at 4 °C overnight, followed by Alexa Fluor 594-conjugated donkey anti-rabbit IgG (Life Technologies Ltd., Paisley, UK) for 2 h. Images were obtained by confocal microscopy (Eclipse TE200-U, Nikon UK Ltd., Surry, UK). The Fiji Image-J Software was used for image analysis.

To measure cell motility and migration, we used an in vitro wound-healing assay. PANC1, MiaPaCa-2, and BxPC-3 cells (4 × 10⁵ cells/well), as well as MDA-MB-231 and MDA-MB-436 cells (2 × 10⁵ cells/well), were plated in six-well plates and cultured in medium containing 10% FBS. After 24 h of incubation, cells were transfected with control miRNA or miR-873 mimic, KRAS siRNA, or control siRNA. The second day after transfection, we used a 200-μL sterile pipette tip to do straight scratch on the confluent cell layers. Then cells were photographed using a phase-contrast microscope (Nikon Eclipse TE-200-U) at 0 h. The cells were continued in incubation, and images were taken again at 24 and 36 h. The wound healing was displayed by comparing photographs taken at 0 h with those taken 24 and 36 h later. At least 5 random non-overlapping pictures for each experiment were tested and quantitated using ImageJ software (NIH, Bethesda, MD).