Samples were fixed in 4% (wt/vol) paraformaldehyde solution (Wako) for 15 min at room temperature on day 3 (for NVCM) and day 7 (for iPSC-CM) following seeding respectively. They were subsequently blocked and permeabilized with 2% (wt/vol) bovine serum albumin (BSA) (Gemini Bio-Products) solution with 0.1% (vol/vol) Triton X-100 (Promega) for 1 h at room temperature. Samples were then incubated with mouse monoclonal antibodies against alpha-actinin (1:100, Sigma) and rabbit monoclonal antibodies against connexin 43 (1:100, Cell Signaling Technology) overnight at 4 °C. Following primary antibody incubation, samples were washed three times with PBS at room temperature, followed by 1 h room temperature incubation with fluorophore-conjugated anti-IgG antibodies (1:200, Biotium). Hoescht 33258 (Invitrogen) nucleus counterstain was also performed. Following 3 times of washing with PBS (Gibco, Life Technologies), fluorescently stained samples were stored in PBS (Gibco, Life Technologies) with 0.05% (wt/vol) sodium azide (Alfa Aesar) at 4 °C and imaged within 1 week of staining.
Paraformaldehyde solution
Manufactured by Fujifilm
Sourced in Japan
The 4% paraformaldehyde solution is a laboratory reagent used for the fixation of biological samples. It is a buffered aqueous solution containing 4% w/v paraformaldehyde, a common fixative. This solution is primarily used for preserving the structural integrity of cells and tissues prior to further analysis or processing.
Automatically generated - may contain errors
Lab products found in correlation
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Dnase 1 solution, by Merck Group (2 mentions)
Triton x 100 solution, by Merck Group (2 mentions)
Tunel solution, by Roche (2 mentions)
Cm3050 cryostat, by Leica (2 mentions)
Oct compound, by Sakura Finetek (2 mentions)
Biorevo bz 9000 microscope, by Keyence (2 mentions)
Sodium azide, by Thermo Fisher Scientific (1 mentions)
Rabbit monoclonal antibodies against connexin 43, by Cell Signaling Technology (1 mentions)
Triton x 100, by Promega (1 mentions)
Fluorophore conjugated anti igg antibodies, by Biotium (1 mentions)
Bovine serum albumin bsa, by Gemini Bio (1 mentions)
Infinite m200 microplate reader, by Tecan (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Midazolam, by Sandoz (1 mentions)
Butorphanol, by Meiji Seika Pharma (1 mentions)
3 3 diaminobenzidine tetrahydrochloride dab, by Agilent Technologies (1 mentions)
Streptavidin horseradish peroxidase hrp, by Agilent Technologies (1 mentions)
Abiorevo bz 9000 microscope, by Keyence (1 mentions)
25 protocols using paraformaldehyde solution
1
Immunofluorescence Staining of Cardiac Cells
2
Autophagy Flux Measurement in HeLa Cells
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Autophagic flux after drug treatment was determined essentially as described previously35 (link). GFP-LC3-RFP-LC3ΔG-expressing HeLa cells were seeded in black/clear bottom 96-well plates (353948, Corning, NY) at 1.5 × 104 cells/well and maintained for 72 h. After drug treatment for 6 h, cells were washed with PBS (+), fixed with 4% paraformaldehyde solution (163–20145, Wako) for 10 min, and washed with PBS (+). Measurement of GFP and RFP fluorescence was performed using a microplate reader (Infinite M200 microplate reader; Tecan, Mannedorf, Switzerland) with excitation/emission at 480/510 nm and 580/610 nm, respectively.
3
Evaluating DNA Fragmentation in Oocytes
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The TUNEL assay and propidium iodide staining were
performed to evaluate DNA fragmentation in oocytes.
Following several washings in PBS (Gibco, Grand Island, NY,
USA), oocytes were fixed in 3.7% paraformaldehyde solution
(Wako, Japan), treated with 0.1% Triton X-100 solution
(Sigma, Germany) for 40 minutes and exposed to the blocking
solution at 4˚C overnight. Then, oocytes were primarily
incubated in TUNEL solution (Roche, Germany) at 37˚C for
an hour according to the manufacturer’s guidelines. Negative
control oocytes were incubated only in the fluorescent solution
lacking the enzyme to ensure the absence of labeling. For the
positive control, a number of oocytes prior to the incubation
with TUNEL staining solution were incubated with 50 µg/ml
of the DNase I solution (Sigma, Germany) for one hour and
then treated with the TUNEL solution. Oocytes were stained
with 50 µg/ml of the propidium iodide (Sigma, Germany)
solution for 20 minutes to label nuclei and examined under a
fluorescent microscope (Labomed, USA).
performed to evaluate DNA fragmentation in oocytes.
Following several washings in PBS (Gibco, Grand Island, NY,
USA), oocytes were fixed in 3.7% paraformaldehyde solution
(Wako, Japan), treated with 0.1% Triton X-100 solution
(Sigma, Germany) for 40 minutes and exposed to the blocking
solution at 4˚C overnight. Then, oocytes were primarily
incubated in TUNEL solution (Roche, Germany) at 37˚C for
an hour according to the manufacturer’s guidelines. Negative
control oocytes were incubated only in the fluorescent solution
lacking the enzyme to ensure the absence of labeling. For the
positive control, a number of oocytes prior to the incubation
with TUNEL staining solution were incubated with 50 µg/ml
of the DNase I solution (Sigma, Germany) for one hour and
then treated with the TUNEL solution. Oocytes were stained
with 50 µg/ml of the propidium iodide (Sigma, Germany)
solution for 20 minutes to label nuclei and examined under a
fluorescent microscope (Labomed, USA).
4
Tissue Extraction and Fixation Protocol
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We treated experimental animals in accordance with the Kurume University guidelines. C57BL/6N mice were purchased from SLC (Japan SLC, Inc., Shizuoka, Japan). Eight-week-old male mice were anaesthetized by intraperitoneal injection of dexmedetomidine (ZENOAQ, Fukushima, Japan), midazolam (Sandoz K.K., Tokyo, Japan) and butorphanol (Meiji Seika Pharma Co., Tokyo, Japan) at 4, 10 and 0.5 mg/kg, respectively, before rapid decapitation with a sharp blade for extraction of mRNA or proteins or before fixation by perfusion with 4% paraformaldehyde solution (PFA: FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for immunohistochemistry.
5
Immunohistological Analysis of H-D Antigen
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A homozygous GalT/CMAH double KO pig (M129-2), a homozygous
GalT KO pig (M71-5) and an age-matched wild-type (WT) pig were sacrificed 2 days after
birth. Harvested samples were used for immunohistological analysis of the H-D antigen. The dissected organs
were fixed in a 4% paraformaldehyde solution (Wako Pure Chemical Industries, Osaka, Japan), embedded in
paraffin, sectioned and stained with hematoxylin using standard methods.
The fixed sections were also incubated with blocking solution (2% BSA/D-PBS) for 1 h and then treated with a
chicken anti-H-D antibody (a generous gift from Prof N Wakamiya, Asahikawa Medical University) for 1 h. After
removal of the excess antibody, the sections were incubated with biotin-conjugated goat anti-chicken IgY
(Abcam, Cambridge, United Kingdom) for 30 min. For detection of the α-Gal antigen, the sections were incubated
for 1 h with biotin-conjugated Griffonia simplicifolia I (GS-IB4) lectin (Life Technologies).
Each section was also incubated with Streptavidin-Horseradish Peroxidase (HRP) (DAKO, Tokyo, Japan) for 15 min
and 3,3′-Diaminobenzidine tetrahydrochloride (DAB) (DAKO) for 5 min. The slides were visualized using a
Biorevo BZ-9000 microscope (Keyence, Osaka, Japan).
GalT KO pig (M71-5) and an age-matched wild-type (WT) pig were sacrificed 2 days after
birth. Harvested samples were used for immunohistological analysis of the H-D antigen. The dissected organs
were fixed in a 4% paraformaldehyde solution (Wako Pure Chemical Industries, Osaka, Japan), embedded in
paraffin, sectioned and stained with hematoxylin using standard methods.
The fixed sections were also incubated with blocking solution (2% BSA/D-PBS) for 1 h and then treated with a
chicken anti-H-D antibody (a generous gift from Prof N Wakamiya, Asahikawa Medical University) for 1 h. After
removal of the excess antibody, the sections were incubated with biotin-conjugated goat anti-chicken IgY
(Abcam, Cambridge, United Kingdom) for 30 min. For detection of the α-Gal antigen, the sections were incubated
for 1 h with biotin-conjugated Griffonia simplicifolia I (GS-IB4) lectin (Life Technologies).
Each section was also incubated with Streptavidin-Horseradish Peroxidase (HRP) (DAKO, Tokyo, Japan) for 15 min
and 3,3′-Diaminobenzidine tetrahydrochloride (DAB) (DAKO) for 5 min. The slides were visualized using a
Biorevo BZ-9000 microscope (Keyence, Osaka, Japan).
6
Kidney Isolation and Preservation
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Blood was collected from the tail vein without anesthesia. Urine was collected for 16 hours in a metabolic cage. Rats were operated on at 5 or 12 weeks after the onset of feeding of each diet, under ether or isoflurane anesthesia. After celiotomy, the kidneys were isolated, weighed, and fixed in 4% paraformaldehyde solution (Wako Pure Chemical Co. Ltd., Osaka, Japan). Part of each kidney was embedded in OCT compound and frozen in cold acetone.
7
HeLa Cell Culture on Titanium Film
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HeLa cells were cultured on an EtOH-sterilized 300 nm Ti film deposited on a 100 µm thick glass substrate for 24 h. The cells were fixed with 4 % (w/v) paraformaldehyde solution (161–20141, Fujifilm Wako, JP) for 15 min at room temperature. After washing with distilled water, the cells were exposed to air.
8
HeLa Cell Peptide Screening Protocol
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HeLa cells were seeded (at a density of 2.2 × 105 cells/dish) in 35 mm polystyrene dish (AGC Techno Glass) and incubated for 24 h. After cell condition became stable, the cells were incubated with 0.1 µM of each peptide and 1% DMSO at 37 °C for 4 h. The cells were washed with PBS three times and collected with 0.25 w/v% trypsin-1 mM EDTA solution (FUJIFILM Wako Pure Chemical). After washing with PBS, the collected cells were fixed with 4% paraformaldehyde solution (FUJIFILM Wako Pure Chemical) for 10 min. The fixed cells were washed with PBS three times and subjected to FACS analysis using FACS Aria III (Becton Dickinson, Franklin Lakes, NJ, USA).
9
Teratoma Formation Assay for Human iPSCs
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For teratoma formation assay, human iPS cells that dissociated into single cells after long-term culture in our culture system were injected directly into the subrenal capsular space of immunodeficient mice (C.B-17/lcr-scid/scidJcl; CLEA Japan, Tokyo, Japan). After 3 months, the teratomas were surgically dissected out of the mice and fixed with 4% paraformaldehyde solution (Wako Pure Chemical Industries). For immunocytochemistry, each fixed teratoma was sectioned into 7-μm slices using a cryostat. All animal experiments in this study were approved in advance by the University of Tokyo and were conducted in accordance with rules of its Institutional Animal Care and Use Committee.
10
Flow Cytometry Analysis of hiPSCs
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To obtain single cells, the hiPSC aggregates were dissociated by Accumax Cell Aggregate Dissociation Medium (Invitrogen) and passed through a 40 μl cell strainer (Corning). Then, 106 cells were fixed with 4% paraformaldehyde solution (FUJIFILM Wako Pure Chemical) in PBS for 30 min at room temperature, followed by permeabilization with 1% Triton X (Sigma–Aldrich) in PBS for another 30 min, at room temperature. After washing with HBSS, the hiPSCs were incubated for 1 hr with monoclonal mouse IgG2a Anti‐Human Oct‐4A Alexa Fluor 488‐conjugated antibody (R&D Systems). The control group was incubated with anti‐mouse IgG isotype control (R&D Systems) at room temperature. The stained cells were washed with HBSS and the flow cytometry analysis performed by Epics XL (Beckman Coulter, CA). The data were analyzed using Expo32 ADC analysis software (Beckman Coulter).
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