Cells were seeded in a 12-well dish onto cover slips and grown over night. The next day, cells were washed twice in PBS and fixed for 10 minutes in 4% formaldehyde/PBS (Sigma). Coverslips were washed in DMEM (Gibco) containing 10 mM HEPES followed by a 10 min incubation. Coverslips were washed in PBS and permeabilised for 3 min in either 0.2% NP-40/PBS or 0.2% Triton X-100/PBS. Coverslips were washed twice in PBS and blocked for 15 min in 3% BSA (Sigma) in PBS. Primary antibody incubation was done for 1-2 h at room temperature at appropriate antibody dilutions in blocking solution. Residual antibody was washed away in 0.2% Tween/PBS (3x10 min). Secondary antibody incubation was done for 30 min at 1:300 antibody dilution in the dark. The same wash steps were repeated, but the first wash contained DAPI (0.5–1 μg in 10 ml, SigmaAldrich). Finally, coverslips were dipped in water, air dried and mounted on slides with Vectashield (Vector Laboratories). Fluorescence signals were analysed on a Deltavision Widefield microscope (GE). Images were deconvolved using the default settings of softWoRx Imaging software and further analysed using OMERO (Allan et al., 2012 (link)).
Formaldehyde pbs
Manufactured by Merck Group
Sourced in United States, Germany
Formaldehyde/PBS is a laboratory reagent used for the fixation and preservation of biological samples. It is a mixture of formaldehyde and phosphate-buffered saline (PBS). Formaldehyde acts as a fixative, preserving the structure and morphology of cells and tissues, while PBS provides a physiologically relevant buffer to maintain the sample's integrity.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Pd l1 10f 9g2, by BioLegend (2 mentions)
Goat anti rat igg h l alexa fluor 568, by Thermo Fisher Scientific (2 mentions)
Glycine, by Merck Group (2 mentions)
Goat serum, by GE Healthcare (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Deltavision wide field microscope, by GE Healthcare (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Hematoxylin and eosin, by Merck Group (1 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin pbs, by Merck Group (1 mentions)
Pbs bsa, by Thermo Fisher Scientific (1 mentions)
Pbs tween, by Merck Group (1 mentions)
Zoe fluorescent cell imager, by Bio-Rad (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Donkey serum, by Merck Group (1 mentions)
12 protocols using formaldehyde pbs
1
Immunofluorescence Microscopy of Adherent Cells
2
Pancreatic Tissue Histological Analysis
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Remaining portion of the pancreas was fixed overnight at 4°C in freshly prepared formaldehyde/PBS (Sigma-Aldrich, St. Louis, MO, USA) (pH 7.4). After fixation, tissue was embedded in paraffin, sectioned, and processed for hematoxylin and eosin (Sigma-Aldrich) staining following standard protocol. Multiple microscopic fields were randomly selected from each treatment group and assessed for leukocyte infiltration, acinar cell necrosis, and vacuolization by a pathologist, who was blinded to the treatments.
3
Immunofluorescent Detection of PD-L1 in Murine Tissue
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Murine sections were fixed using 4% (w/v) formaldehyde-PBS (Sigma) for 10 min and then washed with 3 changes of Tris-buffered saline (TBS) at pH 8.2. Sections were then blocked for 1 h using 10% goat serum (PAA Laboratories, UK), 1% bovine serum albumin (Sigma), 0.3 M glycine (Sigma) and 0.1% Triton X-100 (Sigma) in TBS and washed with 3 changes of TBS. The primary antibody for PD-L1 (10F.9G2) (Biolegend, USA) was next applied and incubated for 1 h. Following washing with 3 changes of TBS, sections were then incubated with Goat anti-rat IgG (H+L) Alexa Fluor® 568 (Invitrogen, UK) at 1:200 working concentration, for 1 h, and finally washed with 3 changes of TBS.
4
Silver Staining for Light Microscopy
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Sections were fixed in 10% w/v formaldehyde-PBS (Sigma) for 10 min followed by immediate washing in ultra high purity (UHP) water for 5 min. The washed sections were placed in a Coplin jar containing 1% silver nitrate solution and incubated for 1 h at room temperature, then washed in UHP water for 5 min. The sections were covered with UHP water and incubated under ultraviolet light for 1h, followed by a dip in 5% sodium thiosulphate solution for 5 min to remove the un-reacted silver. After a final wash in UHP water for 5 min, sections were counter stained with haematoxylin solution for 1 min, washed and dehydrated using three changes of IMS and cleared with three changes of xylene. The sections were coversliped using xylene based DPX.
5
Immunofluorescent Detection of PD-L1 in Murine Tissue
6
Silver Staining for Light Microscopy
7
Metabolic Activity and Morphology of MC3T3-E1 Cells
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This were performed in triplicate. The average metabolic activity of the cells was calculated using: Metabolic activity
Furthermore, actin staining was performed to observe the morphology of the cells cultured with the extracts. The MC3T3-E1 cells (5 × 10 3 cells) were cultured for 7 days on 48-well glass disks in 200 µL of the extracts. The samples were washed with phosphate buffered saline (PBS, Sigma-Aldrich, Germany), fixed using 4% formaldehyde/PBS (Sigma-Aldrich, Germany) for 15 min at room temperature, and permeabilized with 0.5% Triton/PBS at 4 °C for 5 min. Afterwards, 1% bovine serum albumin (BSA)/PBS (Sigma-Aldrich, Germany) was added to each well, followed by 5 min of incubation, the addition of rhodamine phalloidin (1 : 1000 in 1% BSA/PBS, Life Technologies Corp., USA), and incubation at 37 °C for 1 h. The samples were then washed with 0.5% Tween/PBS (Sigma-Aldrich, Germany) three times prior to being mounted on glass slides with Prolong gold (containing 4′,6-diamidino-2phenylindole (DAPI), Life Technologies, USA). The cytoskeleton and cell nuclei were examined using a fluorescence microscope (ZOE fluorescent cell imager, Bio-Rad Laboratories Inc., USA).
Furthermore, actin staining was performed to observe the morphology of the cells cultured with the extracts. The MC3T3-E1 cells (5 × 10 3 cells) were cultured for 7 days on 48-well glass disks in 200 µL of the extracts. The samples were washed with phosphate buffered saline (PBS, Sigma-Aldrich, Germany), fixed using 4% formaldehyde/PBS (Sigma-Aldrich, Germany) for 15 min at room temperature, and permeabilized with 0.5% Triton/PBS at 4 °C for 5 min. Afterwards, 1% bovine serum albumin (BSA)/PBS (Sigma-Aldrich, Germany) was added to each well, followed by 5 min of incubation, the addition of rhodamine phalloidin (1 : 1000 in 1% BSA/PBS, Life Technologies Corp., USA), and incubation at 37 °C for 1 h. The samples were then washed with 0.5% Tween/PBS (Sigma-Aldrich, Germany) three times prior to being mounted on glass slides with Prolong gold (containing 4′,6-diamidino-2phenylindole (DAPI), Life Technologies, USA). The cytoskeleton and cell nuclei were examined using a fluorescence microscope (ZOE fluorescent cell imager, Bio-Rad Laboratories Inc., USA).
8
Myenteric Plexus Immunostaining Protocol
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After imaging, some Wnt1|GCaMP3 tissues were fixed using 4% formaldehyde/PBS (Sigma-Aldrich) for 2 h at RT for post hoc immunostaining. After washing in PBS (3 x 10 min), the tissues were stored in fresh PBS at 4°C until further processing.
The mucosa, SMP and the circular muscle layer were removed from the fixed tissues by microdissection. The resulting myenteric plexus-longitudinal muscle (LMMP) preparations were incubated for 2 h at RT in blocking solution (4% donkey serum (Merck Millipore)/0.5% Triton-X 100 (Sigma) in PBS). Next, the samples were incubated at 4°C overnight with the primary antibodies (Table 2). After washing with PBS (3 x 10 min), the samples were incubated with the secondary antibodies for 2 h at RT (Table 3). Following another set of washes in PBS (3 x 10 min), the samples were mounted using Citifluor mounting medium (Citifluor Ltd.).
Images were obtained using an LSM 780 confocal microscope (Zeiss) fitted with an argon laser (488 nm) and two solid state lasers (405 and 561 nm). Confocal micrographs display maximum projections of confocal z-stacks. Images were adjusted for brightness and contrast. Neuronal cell counts were performed in ImageJ/Fiji.
The mucosa, SMP and the circular muscle layer were removed from the fixed tissues by microdissection. The resulting myenteric plexus-longitudinal muscle (LMMP) preparations were incubated for 2 h at RT in blocking solution (4% donkey serum (Merck Millipore)/0.5% Triton-X 100 (Sigma) in PBS). Next, the samples were incubated at 4°C overnight with the primary antibodies (Table 2). After washing with PBS (3 x 10 min), the samples were incubated with the secondary antibodies for 2 h at RT (Table 3). Following another set of washes in PBS (3 x 10 min), the samples were mounted using Citifluor mounting medium (Citifluor Ltd.).
Images were obtained using an LSM 780 confocal microscope (Zeiss) fitted with an argon laser (488 nm) and two solid state lasers (405 and 561 nm). Confocal micrographs display maximum projections of confocal z-stacks. Images were adjusted for brightness and contrast. Neuronal cell counts were performed in ImageJ/Fiji.
9
Formaldehyde Fixation and Calcein Staining
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Sections were fixed in 2% formaldehyde-PBS (Sigma) for 10 min and then washed in three changes of 0.1 M Tris-HCL buffer, for 3 min each. Fixed sections were incubated with 2.5 μM calcein Tris-HCL solution in a glass coplin jar for 20 min at room temperature and with light exposure restricted to the minimum. Following incubation, the sections were washed carefully with three changes of 0.1 M Tris-HCl buffer for 3 min each.
10
Formaldehyde Fixation and Calcein Staining
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