A lower dose than the reported LD50 for SEA15 (link) was used to activate lymphocytes.18 (link) Induced apoptosis caused by S-SEA and C-SEA was examined by staining with Hoechst 33258 (Wako, Osaka, Japan)19 (link); a total of 1000 lymphocytes were randomly observed under the high-power field (×400 magnification) to evaluate the number of apoptotic cells under different conditions; the ratio of apoptotic cells per 1000 lymphocytes was calculated.20 (link) The lymphocytes exposed to different concentrations of S-SEA and C-SEA were cultured for 72 hours; then the lymphocyte smears were prepared, fixed in 10% formalin, and stained with Hoechst 33258. Finally, the ratios of apoptotic cells were determined using a fluorescent microscope (Figure 3 ). In the next step, the optimal concentrations of S-SEA and C-SEA for generating apoptosis were determined by kinetic curve (1000) and then, this optimal concentration was further studied. The procedure was repeated 3 times (Figure 4 ).
Hoechst 33258
Manufactured by Fujifilm
Sourced in Japan
Hoechst 33258 is a fluorescent dye used in laboratory applications. It specifically binds to adenine-thymine (A-T) rich regions of DNA, emitting fluorescence upon binding. This property makes it a useful tool for DNA detection and quantification.
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Lab products found in correlation
Fluoview fv10i, by Olympus (2 mentions)
Anti calbindin d 28k antibody, by Merck Group (1 mentions)
M o m blocking reagent, by Vector Laboratories (1 mentions)
Las af software, by Leica (1 mentions)
Blocking solution, by Nacalai Tesque (1 mentions)
Alexa fluor 594 donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Glial fibrillary acidic protein (gfap), by Fujifilm (1 mentions)
Ham s f12, by Fujifilm (1 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Microtubule associated protein 2, by Synaptic Systems (1 mentions)
Anti rabbit igg horseradish peroxidase linked whole antibody from donkey, by GE Healthcare (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Ps6 ser235 236, by Cell Signaling Technology (1 mentions)
Lsm800 confocal microscope, by Zeiss (1 mentions)
Ix81 fluorescence microscope, by Olympus (1 mentions)
Bovine serum albumin (bsa), by Fujifilm (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
10 protocols using hoechst 33258
1
Lymphocyte Apoptosis Induction by SEA
2
Immunofluorescent Labeling of Mouse Brain Sections
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For immunofluorescence staining of brain sections, mouse brains were perfusion-fixed with 4% paraformaldehyde, cryoprotected in 30% sucrose solution, frozen in optimal cutting temperature compound (Tissue-Tek), and cut into 10-μm-thick sections. The frozen tissue sections were washed with phosphate-buffered saline (PBS; pH 7.4) and permeabilized in 5% bovine serum albumin/0.5% Triton X-100 in PBS for 30 min. After blocking endogenous biotin with an endogenous avidin-biotin blocking kit (Nichirei) and M.O.M. blocking reagent (Vector Laboratories) according to the manufacturer’s protocol, the sections were incubated overnight with anti-calbindin-D-28K antibody (1:2000; Sigma). Primary antibody binding was detected with M.O.M. biotinylated anti-mouse immunoglobulin G reagent (Vector Laboratories), subsequently stained with streptavidin-conjugated Cy3 (Vector Laboratories). For nuclear staining, Hoechst 33258 (Wako) was used. Fluorescence was visualized using a confocal laser scanning microscope (FluoView FV10i, Olympus).
3
Laser Micro-irradiation Analysis of Nbs1 Proteins
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Laser micro-irradiation analysis was performed as previously described with a few modifications [45 (link),46 (link)]. We seeded 2 × 105 of cultured cells expressing wild-type olNbs1-Venus or olNbs1 (H170)-Venus into the glass-bottom dishes and cultured the cells for 2 days at 33°C. After heat treatment (2 h) at 41°C to induce olNbs1-Venus or olNbs1 (H170)-Venus expression, the cells were incubated overnight and then incubated with 4 μM Hoechst 33258 (Wako, Osaka, Japan) in L15 medium supplemented with 20% FBS for 10 min at 33°C. Then, the cells were washed in L15 medium (phenol red free) before laser irradiation. Images were obtained using a SP6 confocal microscope (Leica Microsystems, Wetzlar, Germany) and analyzed using LAS AF software (Leica Microsystems). We set a strip-shaped region of interest (ROI) and 5 shots of 405 nm laser was irradiated into the ROI, inducing DSBs in the restricted part of the nuclei. Seventeen images were captured at 15 s intervals from 1.3 s to 241.3 s after the micro-irradiation, and then 3 more images were captured at 30 s intervals. The mean fluorescence intensity of the irradiated ROI in the nucleus was digitalized using Image J. Fold increase (F. I.) at each time point was calculated by following equation; F. I. (%) = 100 × ((mean intensity at each time point)–(mean intensity at t = 1.3 s))/(mean intensity at t = 1.3 s).
4
Mouse Brain Immunofluorescence Imaging
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Mouse brains were perfusion-fixed with 4.0% paraformaldehyde (PFA) 4 weeks after cranial window surgery over the primary visual cortex (Fig. 1e, f , Supplementary Fig. 2a, b ). Brains were removed, fixed overnight in 4% PFA, and cut into 50-μm thick coronal sections using a vibratome (7000smz; Campden Instruments, Leicestershire, UK). Slices were then incubated in 10% blocking solution (NACALAI TESQUE, INC., Japan) containing 0.2% Triton X-100 for 30 minutes at room temperature, followed by incubation with a primary antibody against glial fibrillary acidic protein (GFAP, 1:1,000, FUJIFILM Wako Pure Chemical Corporation, Japan) overnight (~16 h) at 4°C. After washing with 0.1% Tween 20 in PBS, slices were incubated with secondary antibody (1:500; Alexa Fluor 594 donkey anti-mouse IgG; Thermo Fisher Scientific, Waltham, MA, USA) for 4 h at room temperature. Nuclei were counterstained with 1:2000 Hoechst 33258 (FUJIFILM Wako Pure Chemical, Japan). Finally, stained slices were examined using a confocal microscope (Leica TCS SP8 STED 3X FALCON, Leica, USA) with an HC PL APO CS2 ×10/0.40 NA dry objective lens. Mean Hoechst 33258 and GFAP fluorescence emission levels were measured at 15 ROIs from three mice in each condition using ImageJ and normalized per 300 × 300 µm2.
5
Neuronal Differentiation of PC12 Cells
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Rat pheochromocytoma PC12 cells were maintained in Dulbecco’s modified Eagle’s medium (Gibco) and Ham’s F-12 (Wako) containing 5% (v/v) fetal bovine serum (FBS) (Cell Culture Technology), 5% horse serum (Gibco), and 1% antibiotic-antimycotic (Gibco) in a 5% CO2 humidified incubator at 37°C. Cells were seeded on a dish (35 mm, ibidi) that had been coated with poly l -lysine at a density of 5 × 103 per dish and were transfected with Lipofectamine LTX (Invitrogen) according to the manufacturer’s protocol. For the differentiation assay, 24 hours after transfection, the cells were cultured with NGF, which was added at a final concentration of 100 ng ml−1 to the serum-free cell culture medium, for 24 hours. The cells were fixed with 3.7% formaldehyde (Wako). Fluorescence images were acquired with identical imaging parameters using a confocal laser scanning microscope (FluoView FV10i, Olympus) and were batch-analyzed using HCA-Vision software (CRISO) through automated neuron body detection, neurite detection, and neurite analysis, with identical parameters. The areas of cell body and neurite outgrowth were visualized with Venus, and the nucleus was stained with Hoechst 33258 (Wako).
6
Immunocytochemistry and Western Blotting Analysis
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The following primary antibodies were used for immunocytochemistry: monoclonal mouse antibody β-Ⅲ tubulin (Tuj-1) (1:1,000; Sigma), polyclonal guinea pig antibody microtubule-associated protein 2 (MAP2) (1:1,000; Synaptic Systems, Goettingen, Germany) and polyclonal rabbit antibody phospho-S6 ribosomal protein (pS6) Ser 235/236 (1:1,000; Cell Signaling Technology, Danvers, MA, United States). The following secondary antibodies were used: CF647 donkey anti-guinea pig IgG (H + L) highly cross-adsorbed (1:1,000; Biotium) and CF555 donkey anti-rabbit IgG (H + L) highly cross-adsorbed (1:1,000; Invitrogen). Nuclei were stained with Hoechst 33,258 (1:1,000; Wako).
The following primary antibodies were used for western blotting: polyclonal rabbit antibody pS6 Ser 235/236 (1:500; Cell Signaling Technology) and monoclonal rabbit antibody β-actin (1:500; Cell Signaling Technology). The following secondary antibodies were used: anti-rabbit IgG horseradish peroxidase linked whole antibody from donkey (1:10,000; GE Healthcare).
The following primary antibodies were used for western blotting: polyclonal rabbit antibody pS6 Ser 235/236 (1:500; Cell Signaling Technology) and monoclonal rabbit antibody β-actin (1:500; Cell Signaling Technology). The following secondary antibodies were used: anti-rabbit IgG horseradish peroxidase linked whole antibody from donkey (1:10,000; GE Healthcare).
7
Immunostaining of HeLa Cells and Neurons
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Immunostaining was performed as described previously55 (link). HeLa cells or primary neurons cultured on glass cover slips were fixed for 10 min at room temperature with 3.7% formaldehyde in phosphate-buffered saline (PBS) and then incubated consecutively with primary antibodies and Alexa Fluor 488- or Alexa Fluor 546-labeled secondary antibodies in PBS containing 0.5% Triton X-100. Nuclei were also stained with Hoechst 33258 (Wako, Tokyo, Japan) as indicated. The cells were finally covered with a drop of GEL/MOUNT (Biomeda, Hayward, CA) and observed with an Olympus IX-81 fluorescence microscope (Olympus, Tokyo, Japan) or LSM800 confocal microscope (Zeiss, Tokyo, Japan).
8
Immunofluorescence Staining for Cytoskeleton and Nuclei
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After the cells were cultured for 24 h after cell seeding, the cells were fixed with PBS containing 4% paraformaldehyde for 10 min and washed with PBS 3 times. Then, the cells were permeabilized by treatment with 1% TritonTM X-100 (Sigma-Aldrich, St. Louis, MO, USA), followed by blocking with 2% bovine serum albumin (BSA, FUJIFILM Wako Pure Chemical, Osaka, Japan) in PBS. After washing with PBS 3 times, the cells were stained with Alexa Fluor-488 phalloidin (dilute in PBS at 1:40, Invitrogen, Waltham, MA, USA) for 20 min at room temperature (avoid light). The stained cells were washed with PBS and stained with Hoechst 33,258 (dilute in PBS at 1:1000, FUJIFILM Wako Pure Chemical, Osaka, Japan) for 10 min to stain cell nuclei. Fluorescence images of the stained cells were obtained using a fluorescence microscope with a DP-70 CCD camera.
9
Immunofluorescence Staining of Induced Cells
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Induced cells were fixed, washed as described above, and blocked with 2% bovine serum albumin (BSA) in PBS for 1 h at room temperature. Then, the cells were stained with the following primary antibody in 2% BSA/PBS overnight at 4 °C: PE-conjugated anti-human CD146 (clone: P1H12) antibody (1:100; BD Biosciences; 550315). After two washes, the cells were stained with Hoechst 33258 (1:1000; FUJIFILM; 343-07961), 1 µg/mL Bodipy 493/503 (Invitrogen; D3922), and Alexa Fluor 555-conjugated anti-mouse IgG1 secondary Antibody (1:1000; Invitrogen; A-21127) for 1 h at room temperature. The stained cells were washed twice and mounted using Fluoremount g (Southern Biotech, Birmingham, AL, USA; 0100-01). The prepared samples were observed under an LSM 700 confocal microscope (Zeiss, Oberkochen, Germany).
10
Immunofluorescence Imaging of Murine Brain
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Mice brain sections were prepared as described above. From bregma to bregma posterior 1.0 mm, the brains were sectioned into 15 μm. The slices were washed with PBS for 30 minutes, blocked with 3% horse serum in 0.1% PBSTr (PBS with 0.01% Triton X-100) for 10 minutes, 3% horse serum in 0.01% PBSTr for 30 minutes and incubated with anti-Hbα rabbit monoclonal IgG (1:50, Cat# SN70-09, Invitrogen, Carlsbad, CA, USA) overnight at 4°C. Slides were washed three times for 10 minutes each with 0.01% PBSTr the following day and incubated with Alexa Fluor 594 (1:1000, Abcam, Tokyo, Japan) rabbit IgG for 1 hour at room temperature. Following 3 times of 0.01% PBSTr washing, slices were incubated for 30 min with Hoechst 33258 (Fujifilm, Tokyo, Japan). Subsequently, the sections were washed with 0.01% PBSTr three times and mounted with Fluoromount™ aqueous mounting medium (MERCK). Images were acquired by confocal laser scanning microscope in 1.5 µm Z-stacks (FV1000, Olympus).
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