Paraformaldehyde (pfa)

Manufactured by Fujifilm

Sourced in Japan, United States

Paraformaldehyde is a white, crystalline solid that is commonly used in laboratory settings. It is a polymer of formaldehyde and serves as a primary fixative in the preparation of biological samples for microscopy and histological analysis.

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Lab products found in correlation

Triton x 100, by Merck Group (31 mentions) Bovine serum albumin (bsa), by Merck Group (28 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (20 mentions) 4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (19 mentions) Hoechst 33342, by Thermo Fisher Scientific (18 mentions) Alexa fluor 488, by Thermo Fisher Scientific (14 mentions) Oct compound, by Sakura Finetek (13 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (12 mentions) Triton x 100, by Fujifilm (10 mentions) Lsm 710, by Zeiss (10 mentions) Bz 9000, by Keyence (9 mentions) Lsm 780, by Zeiss (9 mentions) Lsm 880, by Zeiss (8 mentions) Lsm 700, by Zeiss (8 mentions) Bz x700, by Keyence (6 mentions) Bz x710, by Keyence (6 mentions) Rhodamine phalloidin, by Thermo Fisher Scientific (6 mentions) Bz 2 analyzer software, by Keyence (5 mentions) Hoechst 33342, by Dojindo Laboratories (5 mentions) Alizarin red s, by Merck Group (5 mentions)

422 protocols using paraformaldehyde (pfa)

Pluripotency Marker Staining in iPSCs

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Alkaline phosphatase staining was performed according to the manufacturer's instructions. Briefly, the iPSCs were fixed with 4% paraformaldehyde (Fujifilm Wako, Osaka, Japan) for 3 min and incubated with the staining solution for 15 min in the dark. The stained iPSC colonies were examined using bright-field microscopy.
For immunofluorescence staining, the iPSCs were fixed with 4% paraformaldehyde (Wako) for 20 min and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich. St. Louis, MO, USA) for 10 min. The cells were blocked for 30 min in 1% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) and incubated with primary antibodies. The cells were treated with the secondary antibodies for 1 h; counterstaining was performed with 4 ′ ,6-diamidino-2-phenylindole (DAPI). The images were analyzed using fluorescence microscopy (Olympus JP/IX83, Tokyo, Japan).

Kim Y., Yun B., Ye B.S, & Kim B.Y. (2024). Generation of Alzheimer's Disease Model Derived from Induced Pluripotent Stem Cells with APP Gene Mutation. Biomedicines, 12(6).

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Tracking Neuronal Activity in Mice

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We acquired the data using a 32-channel Digital Lynx SX acquisition system (Neuralynx), sampled signals at 32 KHz. We placed the stainless-steel electrodes to screws affixed on the skull above the cerebellum as a ground and tetrodes in the corpus callosum as a reference. A pair of red/green light emitting diodes affixed to the microdrive allowed us to track the position and head direction of animals. At the conclusion of the experiment, we anesthetized mice with avertin and electrode positions were marked by electrolytic lesioning of brain tissue (with 50-μA current for 10–12 s through each tetrode individually). Mice were transcardially perfused with phosphate-buffered saline followed by 4% PFA (Wako chemicals), after which brains were removed and post-fixed for a further 24 h in 4% PFA. Coronal slices 40 μm thick were prepared on a vibratome (Leica), stained with DAPI (Vectashield) and signals were imaged by epifluroscence microscopy (DM6000B, Leica) to confirm electrode placement and reporter expression when appropriate.

Adaikkan C., Joseph J., Foustoukos G., Wang J., Polygalov D., Boehringer R., Middleton S.J., Huang A.J., Tsai L.H, & McHugh T.J. (2024). Silencing CA1 pyramidal cells output reveals the role of feedback inhibition in hippocampal oscillations. Nature Communications, 15, 2190.

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Histological Analysis of WAT and Liver

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WAT and liver tissues were fixed with 4% paraformaldehyde (PFA) (Wako Pure Chemical) for 24 h prior to dehydration and paraffin embedding, followed by cutting into 3 μm sections for liver and 5 μm sections for WAT. Those sections were stained with hematoxylin and eosin to evaluate their structural differences. Keyence BZ-X800 microscope (Keyence) was used to capture images of those sections.

Prabata A., Ikeda K., Rahardini E.P., Hirata K.I, & Emoto N. (2021). GPNMB plays a protective role against obesity-related metabolic disorders by reducing macrophage inflammatory capacity. The Journal of Biological Chemistry, 297(5), 101232.

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Immunohistochemical Analysis of ALK5 and FOXO1 in Mouse Knee Joints

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Knee joints from mice were fixed with 4% paraformaldehyde (PFA) (Wako Pure Chemical Industries) for 2 days, decalcified with K-CX (FALMA, Tokyo, Japan) for 2 days, and embedded in paraffin. Sections were cut into 4 μm–thick sections, deparaffinized, and rehydrated; antigen retrieval was performed by incubation overnight with ethylenediaminetetraacetic acid (EDTA) (1 mM) at pH 8.0. Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxidase (H₂O₂) in methanol for 30 minutes. After blocking with normal horse serum (Vectastain Universal Elite ABC kit; Vector Laboratories, Burlingame, CA, USA) for 30 minutes, sections were incubated with antibodies against ALK5 (sc-398; Santa Cruz Biotechnology), FOXO1 (#2880; Cell Signaling Technology) and normal rabbit IgG (AB-105-C; R&D Systems, Minneapolis, MN, USA) for 1 hour. Sections were incubated with biotinylated secondary antibodies for 30 minutes, followed by incubation with streptavidin–peroxidase complex (Vectastain Universal Elite ABC kit) for 30 minutes. Antibody complexes were visualized using the diaminobenzidine substrate system (Wako Pure Chemical Industries), and counterstained with hematoxylin. The percentage of cells positive for ALK5 and FOXO1 was determined using the BZ-II Analyzer software (Keyence, Osaka, Japan).

Kurakazu I., Akasaki Y., Tsushima H., Sueishi T., Toya M., Kuwahara M., Uchida T., Lotz M.K, & Nakashima Y. (2021). TGFβ1 signaling protects chondrocytes against oxidative stress via FOXO1–autophagy axis. Osteoarthritis and cartilage, 29(11), 1600-1613.

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Histological and Immunohistochemical Analysis of Distal Colon

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For histological analysis, 0.5 cm distal colon was fixed in Zinc Formalin (Polyscience Inc.) for 3 hours and then embedded in paraffin blocks. We then prepared 5 μm paraffin cross-sections, which were used for hematoxylin and eosin (H&E) staining (Mayer’s Hematoxylin solution, 1% Eosin Solution, Wako) following standard procedures. The histological images were captured using the BX51-P Polarizing microscope (Olympus) and processed with the Olympus D.P. Controller 2002 software.
For immunohistochemistry analysis, 0.5 cm distal colon was fixed overnight in 4% paraformaldehyde (PFA) (Wako) at 4°C and mounted in embedding medium Tissue-Tek O.C.T Compound (Sakura). The tissues were cut into 8 μm sections and permeabilized with 0.2% saponin (Nacalai Tesque) in PBS. The sections were then blocked with 5% goat serum (Wako) for Mucin 2 detection. Subsequently, the sections were stained with anti-Mucin2 (1:200, rabbit, clone: H-300, Santa Cruz Biotechnology) at 4°C overnight. For the second antibody, Alexa Fluor 488 conjugated donkey anti-rabbit IgG antibody (1:400, Thermo Fisher Scientific) was used with DAPI (1:1000, Dojindo). The sections were assessed using the Leica TCS SP8 (Leica Microsystems).

Shi Z., Takeuchi T., Nakanishi Y., Kato T., Beck K., Nagata R., Kageyama T., Ito A., Ohno H, & Satoh-Takayama N. (2022). A Japanese Herbal Formula, Daikenchuto, Alleviates Experimental Colitis by Reshaping Microbial Profiles and Enhancing Group 3 Innate Lymphoid Cells. Frontiers in Immunology, 13, 903459.

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Immunocytochemistry Protocol for Cell Labeling

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The antibodies used for ICC are listed in the table below. Cells were fixed in chilled methanol (−30°C) on ice for 5 min. In some experiments, cells were fixed in 4% paraformaldehyde (PFA) (Wako, Osaka, Japan) at room temperature for 15 min and permeabilized by treatment with PBS containing 0.05% Triton X-100 for 15 min. Thereafter, cells were washed three times with PBS, incubated in Blocking One solution (Nacalai Tesque, Kyoto, Japan) at 4°C for 30 min, and labeled with primary antibodies at room temperature for 1 hr or at 4°C overnight. The primary antibodies were detected using Alexa Fluor 488- or Alexa Fluor 594-conjugated secondary antibodies (Life Technologies). Nuclei were counterstained with Hoechst 33342 (Dojindo).

Katsuda T., Matsuzaki J., Yamaguchi T., Yamada Y., Prieto-Vila M., Hosaka K., Takeuchi A., Saito Y, & Ochiya T. (2019). Generation of human hepatic progenitor cells with regenerative and metabolic capacities from primary hepatocytes. eLife, 8, e47313.

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Immunofluorescent Staining of βIII Tubulin

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Cells were mounted on slides, washed twice with PBS, and fixed with 4%
paraformaldehyde (163-20145; FUJIFILM) at 4 °C for 30 min. For frozen
sections, slides were reheated and dried at room temperature for 30
min and then washed in PBS for 5 min to remove the residual O.C.T.
Compound (4583; Sakura Finetek Japan Co., Ltd., Tokyo, Japan). After
permeabilization with 0.1% Triton X-100 (HFH10; Thermo Fisher
Scientific) for 5 min at room temperature, all slides were incubated
with 3% bovine serum albumin (BSA) diluted from MACS BSA Stock
Solution (130-091-376; Miltenyibiotec, Bergisch Gladbach, Germany) in
PBS for 60 min at room temperature. Then, the slides were incubated
with an anti-βIII tubulin antibody (ab18207, 1:500; Abcam, Cambridge,
UK) at 4 °C overnight. The next day, the slides were washed three
times with PBS and incubated with Alexa Fluor 488-conjugated secondary
antibodies (A-11008, 1:1000; Thermo Fisher Scientific, Inc.) for 1 h
at 37 °C in the dark. After three washes with PBS in the dark, ProLong
Gold Antifade Mountant with DAPI (P36931; Thermo Fisher Scientific)
was used to stain the nuclei for 10 min in the dark. The slides were
observed and photographed under a fluorescence microscope (BZ-X700;
KEYENCE, Tokyo, Japan). For negative controls, the primary antibody
was replaced with BSA.

Chen S., Ikemoto T., Tokunaga T., Okikawa S., Miyazaki K., Yamada S., Saito Y., Morine Y, & Shimada M. (2022). Newly Generated 3D Schwann-Like Cell Spheroids From Human Adipose-Derived Stem Cells Using a Modified Protocol. Cell Transplantation, 31, 09636897221093312.

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Fluorescence Imaging of HeLa Cells

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HeLa cells (1 × 10⁴ cells/well) were seeded onto a chambered coverglass (Thermo Fisher, Massachusetts, US) and treated with SAF-Hex at the IC50 concentration for 48 h. Cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde (Fujifilm, Tokyo, Japan) for 15 min at room temperature, and permeabilized with 0.05% Triton X-100 (Fujifilm, Tokyo, Japan). Subsequently, cells were incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 15 min and washed with PBS. Finally, fluorescence images were examined using the FLUOVIEW FV10i confocal system (Olympus, Tokyo, Japan).

Le-Trung N., Duong T.M., Dang T.T, & Kamei K. (2023). Potent anti-cancer activity of Sphaerocoryne affinis fruit against cervical cancer HeLa cells via inhibition of cell proliferation and induction of apoptosis. BMC Complementary Medicine and Therapies, 23, 290.

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Histological Analysis of Mouse Dorsal Skin

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Dorsal skin was excised and fixed with 20% formalin solution (Fujifilm Wako Pure Chemical Corporation). The skin samples were embedded in paraffin by a conventional method and stained with hematoxylin and eosin. Images were acquired using an Olympus virtual slide system VS110 (Olympus, Tokyo, Japan). The thickness of the epidermis was expressed as the mean values from five fields per mouse.
For immunohistochemistry, cryosections of mouse dorsal skin were fixed with 4% paraformaldehyde (Fujifilm Wako Pure Chemical Corporation) for 10 min, washed with 0.1% Triton X-100 in phosphate-buffered saline for 10 min, and incubated with Blocking One (Nacalai Tesque, Kyoto, Japan) for 30 min. Following overnight incubation at 4 °C with primary antibodies, the sections were incubated with Alexa Fluor-conjugated secondary antibodies at room temperature for 60 min in the dark and mounted in PLUS Antifade Mounting Medium (Vector Laboratories, Newark, CA, USA). Antibodies were diluted with Signal Enhancer HIKARI (Nacalai Tesque). Images were acquired using an LSM 700 confocal laser microscope (Carl Zeiss Co., Ltd., Oberkochen, Germany).

Umehara Y., Trujillo-Paez J.V., Yue H., Peng G., Nguyen H.L., Okumura K., Ogawa H, & Niyonsaba F. (2023). Calcitriol, an Active Form of Vitamin D3, Mitigates Skin Barrier Dysfunction in Atopic Dermatitis NC/Nga Mice. International Journal of Molecular Sciences, 24(11), 9347.

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Electron Microscopy of Extracellular Vesicles

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Cells were treated with LEVs or SEVs and incubated for 48 h. Treated cells were fixed by incubating with 200 mM cacodylate buffer containing 8% glutaraldehyde and 20% paraformaldehyde (Wako). After dehydration with ethanol, ultrathin sections were prepared using a Leica EM UC7 microtome (Leica) and collected on 200 mesh copper grids. The sections were stained with 1% uranyl acetate and lead citrate, and images were obtained with a JEOL JEM 1010 transmission electron microscope (JEOL) at 80 kV.

Lee R., Ko H.J., Kim K., Sohn Y., Min S.Y., Kim J.A., Na D, & Yeon J.H. (2019). Anti-melanogenic effects of extracellular vesicles derived from plant leaves and stems in mouse melanoma cells and human healthy skin. Journal of Extracellular Vesicles, 9(1), 1703480.

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