At a predetermined time point, after anaesthesia, the rat diaphragm was cut, and the pericardium was opened. After blood collection from the left apex, 5 mm pieces of tissue from the hypothalamus, adrenal glands, spleen and T3 spinal cord were quickly collected from rats in each group, placed in cryopreservation tubes, frozen in liquid nitrogen, and stored in a low-temperature freezer at − 80 °C. Other rats were perfused with 150 mL of sterile 0.9% normal saline and 300 mL of 4% paraformaldehyde (Biosharp, Beijing, China) after blood collection, and the tissues mentioned above were placed in paraformaldehyde overnight. After dehydration in xylene and an alcohol gradient and embedding in paraffin, the samples were cut into 5 µm continuous sections with a microtome or incubated in a sucrose gradient (10%, 20%, and 30%), embedded in OCT compound, and cut into 20 µm continuous frozen sections with a microtome (Leica, Germany).
Paraformaldehyde (pfa)
Manufactured by Biosharp
Sourced in China, United States
Paraformaldehyde is a solid polymer of formaldehyde. It is a white, crystalline solid that is commonly used as a fixative and preservative in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Solarbio (9 mentions)
Crystal violet, by Solarbio (9 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (9 mentions)
Matrigel, by BD (7 mentions)
Triton x 100, by Merck Group (7 mentions)
Matrigel, by Corning (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (6 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (6 mentions)
Oil red o, by Merck Group (5 mentions)
Transwell chamber, by Corning (5 mentions)
Crystal violet solution, by Solarbio (5 mentions)
Lsm 880, by Zeiss (5 mentions)
Inverted microscope, by Olympus (4 mentions)
Fluorescence microscope, by Olympus (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Lsm 900 confocal microscope, by Zeiss (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Triton x 100, by Beyotime (3 mentions)
Fluorescence microscope, by Leica camera (3 mentions)
220 protocols using paraformaldehyde (pfa)
1
Tissue Harvesting and Preservation Protocol
2
In Vitro Analysis of Composite Hydrogel Scaffold Effects
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Cells used in the vitro experiments were divided into three groups: Control group (medium only), Scaffold group (medium + drug-free composite hydrogel scaffold), and WAY-316606 + Scaffold group (medium + WAY-316606-loaded composite hydrogel scaffold). Cells were fixed with 4% paraformaldehyde (Biosharp, Hefei, China) for 20 min, whereas the spinal cord was not fixed with paraformaldehyde. Following washing with PBS, the cells or tissues were blocked with 10% goat serum for 1 h, washed three times for 5 min with PBS, incubated with primary antibody for 12 h at 4 °C and washed three times for 5 min in PBS. The cells or tissues were subsequently incubated with the corresponding secondary antibody at room temperature for 1 h and washed three times for 5 min in PBS. Finally, 2-(4-)-6-indolecarbamidine dihydrochloride (Sigma) staining was performed. A cover glass was used to seal the spinal cord tissue. Confocal images were obtained using a Leica LSM 800 confocal microscope (Leica, Wetzlar, Germany).
3
Cell Migration and Invasion Assay
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Cells (5 × 104 cells/well for migration assays or 2 × 105 cells/well for invasion assays) were seeded in 200 μl serum-free media into the upper chambers, which were covered with (invasion assays) or without (migration assays) 40 μl Matrigel (BD, New Jersey, USA). The lower chambers contained 600 μl medium supplemented with antibiotics and 10% FBS. After 24 h, the cells that had invaded or migrated were fixed with 4% PFA (Biosharp) for 20 minutes, and the cells in the lower chambers were stained with 0.4% crystal violet for 20 minutes. Finally, the cells were washed with PBS and counted using an inverted microscope (Olympus Corporation; magnification, 200x) with the image processing program ImageJ software version 1.8 (National Institute of Health). Images from 3 random fields of view were captured for each group for analysis.
4
Immunofluorescence Staining of Taste Receptor Cells
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The isolated taste cells were plated onto 10 mm coverslips in Tyrode’s solution for 3–7 h. Then cells were fixed with 4% paraformaldehyde (PFA, Biosharp) in phosphate buffered saline (PBS, Corning) for 15 min and washed three times with PBS. Cells were permeabilized in 0.2% Triton X-100 (Solarbio) in PBS for 20 min at room temperature and washed three times with PBS. Then the cells were blocked for 30 min in 2% BSA (BioFroxx) in PBS. After the above treatment, cells were incubated in primary antibody (1:500, OTOP1 antibody, orb185690, Biorbyt; 1:200, PKD2L1 antibody, orb352708, Biorbyt) at 4 °C overnight. After extensive washing in PBS, Cells were mounted with Aqua-Poly/Mount (18606-20, Polysciences). Images were acquired on an Olympus Flouview 1000 confocal microscope. Related to Fig. 4a .
5
Immunofluorescence Staining of Adherent Cells
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Cells grown on poly-l-lysine coated coverslips were washed with PBS twice and fixed with 4% PFA (Biosharp) at room temperature for 15 minutes. Coverslips were washed with PBS 3 times and then blocked with buffer (5% FBS and 0.2% Triton X-100 in PBS) for 30 minutes, and then incubated with primary antibodies diluted in blocking buffer for overnight at 4°C. On the next day, coverslips were washed with PBS 3 times and incubated with fluorescent secondary antibodies and DAPI (4083; CST) for 1 hour at room temperature. Fluorescence was visualized by LSM900 confocal microscope (Zeiss) using a 63 × oil objective. Co-localization analysis was performed using the Pearson correlation coefficient method in Fiji.
The following antibodies were used for immunofluorescence: rat anti-HA (3F10; Roche), mouse anti-FLAG (F1804; Sigma), chicken anti-GFP (A10262; ThermoFisher), rabbit anti-GM130 (52648; Abcam), Alexa fluor 488 donkey anti-chicken (A78948; ThermoFisher), Alexa fluor 633 goat anti-rat (A-21094; ThermoFisher), Alexa fluor 594 donkey anti-rabbit (R37119; ThermoFisher), and Alexa fluor 488 donkey anti-mouse (A-21202; ThermoFisher).
The following antibodies were used for immunofluorescence: rat anti-HA (3F10; Roche), mouse anti-FLAG (F1804; Sigma), chicken anti-GFP (A10262; ThermoFisher), rabbit anti-GM130 (52648; Abcam), Alexa fluor 488 donkey anti-chicken (A78948; ThermoFisher), Alexa fluor 633 goat anti-rat (A-21094; ThermoFisher), Alexa fluor 594 donkey anti-rabbit (R37119; ThermoFisher), and Alexa fluor 488 donkey anti-mouse (A-21202; ThermoFisher).
6
Colonic Evaluation in Mice Model
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Weight of mice, fecal properties and occult blood were observed daily. Fecal occult blood was detected using occult blood reagent (OB, Leagene, 1216A20). Three weeks later, we withdrew blood from eye frames of mice, before 12 min of centrifugation at 4 °C and 3000×g to obtain serum. Afterwards, all the mice were executed by cervical dislocation method, wherein the whole colon was removed, prior to measurement of colonic length of mice (in each group) and photographing. Part of the colon was stored in the refrigerator at −80 °C, while others was fixed with 4% paraformaldehyde (PFA, Biosharp, 21351381) to prepare the following experiment (Huang et al., 2021 (link)). Later, we collected the feces in the intestine before frozen (at −80 °C) for 16S-rDNA sequencing at a later stage.
7
Immunofluorescence Staining of NPR-B in RAW 264.7 Cells
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RAW 264.7 cell suspensions were plated onto glass coverslips in six-well plates. After 15 h, the glass coverslips were fixed with 4% PFA (Biosharp, Beijing, BL539A) for 20 min and washed with PBS three times for 5 min each time. The autofluorescence quenching agent was added for 5 min. Then the slides were rinsed for 10 min and incubated with 3% hydrogen peroxide for 30 min. After that, the slides were incubated with anti-NPR-B antibody (Santa, 1:200) overnight at 4°C followed by incubation with a secondary FITC Rabbit Anti-goat IgG for 50 min at room temperature. Nuclei were stained with DAPI for 10min.
8
Histopathological Analysis of SARS-CoV-2 Infection
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We followed the same histological approach as described in our previous studies.43 (link)44 (link) In brief, lung tissues form SARS-CoV-2-infected mice were fixed in 4% PFA (Biosharp) for at least 7 d, and then were paraffin embedded (Thermo Fisher Scientific) and cut into 3-μm sections following the standard procedure (RWD Life Science). The tissue slides were stained with H&E (Solarbio) and imaged by the Leica DM 6B light microscopy. The pathological score was evaluated based on the degree of lung tissue lesions including alveolar septal thickening, hemorrhage, inflammatory cell infiltration, and consolidation. The semiquantitative assessment was performed as described previously.47 (link)
9
Histological Analysis of Rat Eyeballs
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On the 14th postoperative day, the eyeballs of rats in the normal, control, and resveratrol groups were removed and placed in 4% paraformaldehyde (PFA; Biosharp, Hefei, Anhui, China) solution at 4°C overnight. After dehydration and paraffin embedding, sections approximately 4 μm thick were cut. After dewaxing and hydration, the sections were stained with hematoxylin and eosin (H&E; Servicebio, Wuhan, Hubei, China). Histological analysis was performed using an inverted microscope (Ts2FL; Nikon, Japan) and CapStudio software.
10
Osteogenic Differentiation of hPDLFs
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5 × 104 hPDLFs were seeded into 12-well plates. When the density reached 70%, the hPDLFs were cultured in osteogenic differentiation medium for 7 days or 21 days, and the hPDLFs were treated in different groups every 2 days. Cells were fixed with 4% PFA (Biosharp, Beijing, China) and stained with BCIP/NBT ALP kit (Beyotime, Shanghai, China) after 7 days of medication. After 21 days of medication, cells were fixed with 4% PFA and stained with 2% Alizarin Red (Solaribo, Beijing, China). Quantify and process data using ImageJ. Each group was set up with 3 duplicate holes, and each experiment was repeated three times.
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