Lsm700 imaging system

Protocol has been described before39 (link). For High-Content Quantitative Imaging (HCQI), photographs were captured on Olympus IX-81 motorized fluorescence microscope equipped with an Olympus UPLSAPO 20x/0.75 NA objective. Automated and unbiased image analysis was carried out with the ScanR acquisition software. For confocal fluorescence microscopy, images were captured on a Zeiss LSM 700 imaging system.

Kidney sections were deparaffinized and hydrated. Heat-mediated antigen retrieval was performed with 10 mM citrate buffer pH 6.0 (Thermo Scientific, IL, USA). Unspecific antigens were blocked by incubating sections for 1 h with 5% goat serum (VE-S-1000, Vector Laboratories). The sections were stained with a Rabbit anti-LRP2 (ab76969, Abcam, 1:1000) antibody, followed by incubation with a Goat anti-Rabbit-AF488 antibody (ab150077, Abcam, 1:500). Sections were mounted with a mounting medium with DAPI (H-1200, Vector) and photographed using the LSM 700 imaging system (Zeiss). The relative fluorescent intensity (RFI) was measured using the ImageJ software (NIH, Bethesda, MD).

Mice were deeply anesthetized by i.p. injection of pentobarbital and perfused transcardially with ice-cold PBS followed by 50 mL ice-cold 4% paraformaldehyde/PBS. The C3 to C5 spinal cord and C3 DRG were quickly removed, post-fixed in the same fixative for 3 hr at 4 °C, placed in 30% sucrose solution for 48 hr at 4 °C, and stored at −80 °C until use. Tissues were embedded in O.C.T compound (Sakura Finetek Japan, Tokyo, Japan) and made at a slice thickness of 14 μm. Fluorescent in situ hybridization (ACDbio, CA, USA) was performed following the manufacturer’s instructions for frozen tissue. Using probes were listed below. Probes: Mm-Grpr (ACDbio, 317871, CA, USA), Mm-Nptx2 (ACDbio, 316901, CA, USA). Tissue sections were analyzed using an LSM700 Imaging System (Carl Zeiss, Oberkochen, Germany).

Pancreas tissues were fixed in buffered 4% formalin for 48 h and then embedded in paraffin. Sections were deparaffinized and hydrated. Heat-mediated antigen retrieval was performed with 10 mM citrate buffer pH 6.0 (Thermo Fisher Scientific, Waltham, MA, USA). Endogenous peroxide was inhibited by incubating with a freshly prepared 3% H₂O₂ solution in MeOH. Unspecific antigens were blockaded by incubating sections for 1 h with 2.5% horse serum (VE-S-2000, Vector Laboratories Inc., Burlingame, CA, USA). For assessing the cellular structure, pancreas sections were stained with guinea pig anti-insulin (A0564, Agilent Dako, Santa Clara, CA, USA; Table 3) antibody, followed by biotinylated secondary antibody and VECTASTAIN ABC reagent (VECTASTAIN ABC-Peroxidase kit, Vector laboratories). Color was developed after incubation with 3,3′-diaminobenzidine (DAB) substrate (ImmPACT DAB peroxidase (HRP) substrate, SK-4105, Vector Laboratories Inc., Burlingame, CA, USA), followed by hematoxylin counterstaining and mounting (Vecmount H-5000, Vector laboratories Inc., Burlingame, CA, USA). Stained sections were photographed using the LSM 700 imaging system (Zeiss, Oberkochen, Germany). Panoramic images were taken for the entire section using ZEN BLUE software (Zeiss, Oberkochen, Germany).

Stable CHIKV cells, stained with BG-TMR-Star with the live-cell protocol, were used for experiments imaging SNAP-nsP3. An LSM700 imaging system (Zeiss) was used for FRAP experiments. Circular bleach areas were drawn within the Zen Black software (diameter of about 0.8 μm). Analyzed ROIs were pooled from recordings of 10 FOVs. One reference region of identical size was drawn over a granule and left unbleached. Another reference region was drawn within the cytoplasm to measure fluorescence background. Bleaching was set with the 405-nm, 488-nm, and 555-nm laser lines at 100% output. Bleaching was started after 3 frames, and another 97 frames were taken every 320 ms during the recovery period. Values of mean ROI intensities were extracted with Zen Black software, exported to Microsoft Excel, and graphed with GraphPad Prism. To induce genuine stress granules in HuH-7 cells, the plasmid pEGFP-G3BP (kindly provided by Richard Lloyd, Baylor University), encoding an EGFP-G3BP1 fusion protein (85 (link)), was transfected with Lipofectamine 2000 reagent (Thermo Fischer Scientific) in cells plated in a 35-mm glass (no. 1.5)-bottom dish with a 27-mm viewing area (Nunc). After 24 h, cells containing G3BP1 granules were identified by live-cell microscopy on an LSM700 confocal system set to 37°C.

According to methods of our previous papers [21 (link), 38 (link)], postnatal mice were deeply anesthetized by pentobarbital and perfused transcardially with PBS, followed by ice-cold 4% paraformaldehyde (PFA)/PBS. Postnatal brain and spinal cord were fixed for 4–6 h and 3–4 h, respectively, in 4% PFA at 4 ℃. Embryonic brains and spinal cord were isolated from E14.5 mice without transcardial PBS perfusion and were immersion fixed for 6 h in 4% PFA at 4 ℃. Embryo and postnatal tissues were placed in 30% sucrose for 48 h at 4 °C and embedded in Tissue-Tek OCT compound (Sakura Finetek, Japan). Cryosections were cut at a thickness of 20 μm, blocked with PBS containing 5% bovine serum albumin and 1% normal donkey serum, and then permeabilized with 0.5% Triton X-100 in blocking solution. The primary antibodies IBA1 (1:1000; 234 004, Synaptic systems, Goettingen, German) and GFP (1:1000; 598, MBL Life science, Tokyo, Japan) were added for 48 h at 4 °C. Tissue sections were incubated with secondary antibodies conjugated to Alexa Fluor 488 (1:1000; A-21,206, Thermo Fisher Scientific, Waltham, MA) and 546 (1:1000; 706-165-148, Jackson immunoReseach LABORATORIES INC., West Grove, Pennsylvania) and mounted with ProLong Glass Antifade Mountant (Thermo Fisher Scientific, Waltham, MA). Tissue sections were analyzed using LSM700 Imaging System (ZEN 2012, Carl Zeiss).