Cells were seeded to be 60 % confluent on glass coverslips in 6-well plates. Cells were washed with PBS and were fixed in 4 % PFA (Sigma) and permeabilized for 10 min in 0.2 % Triton X-100/PBS. Afterwards, samples were washed with 0.1 % Triton X-100/PBS (Roth) and PBS. Subsequently, cells were quenched in 100 mM glycerol/PBS (Roth) and blocked in 1 % BSA/PBS (Serva) for 30 min. Then, cells were incubated with primary antibody (MITF, see above) diluted 1:500 in 1 % BSA/PBS for 1 h. After washing with PBS, coverslips were incubated while covered at room temperature with the secondary antibody AlexaFluor594 goat anti-mouse IgG or AlexaFluor594 goat anti-rabbit IgG (Invitrogen) (1:500 in 1 % BSA/PBS), respectively, for 1 h. After washing with PBS, nuclear counterstaining was performed by incubating with Hoechst 34580 (Invitrogen) (1:10000 in PBS) for 10 min. Afterwards, samples were washed with PBS and distilled water and coverslips were embedded with Mowiol (Sigma) on object slides. Samples were determined by inverted fluorescent microscopy.
Pbs bsa
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
PBS/BSA is a buffered solution that contains phosphate-buffered saline (PBS) and bovine serum albumin (BSA). It is commonly used as a blocking agent in various immunoassays and biochemical techniques to reduce non-specific binding of antibodies or proteins.
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Lab products found in correlation
Ficoll paque plus, by GE Healthcare (8 mentions)
S monovette k3 edta blood collection tubes, by Sarstedt (5 mentions)
Penicillin streptomycin glutamine (psg), by Merck Group (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Alexa fluor 647 phalloidin, by Thermo Fisher Scientific (3 mentions)
Triton x 100, by Thermo Fisher Scientific (3 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (3 mentions)
Bovine serum albumin pbs, by Merck Group (2 mentions)
Pbs tween, by Merck Group (2 mentions)
Zoe fluorescent cell imager, by Bio-Rad (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (2 mentions)
Bsa pbs, by BioLegend (2 mentions)
Tcs sp8, by Leica (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Fluorescent mounting medium, by Agilent Technologies (2 mentions)
C5810 colour intensified 3ccd camera, by Hamamatsu Photonics (2 mentions)
Eclipse 80i microscope, by Nikon (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Triton pbs, by Merck Group (1 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)
30 protocols using pbs bsa
1
Immunofluorescence Staining of MITF
2
Immunofluorescent Staining for Protein Expression
3
Osteoclast Cytoskeletal Dynamics Modulation
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F-actin formation was conducted. RANKL (50 ng/ml) as well as PNC at varying concentrations (0, 31.2, 62.5, and 125 μM) were used to treat RAW 264.7 cells, which were fixed for 15 min in paraformaldehyde (4%), and placed in 0.1% Triton X-100 (Sigma-Aldrich) for 5 min at room temperature after RAW264.7 cell differentiation. We cultured the cells in Alexa Fluor 647 phalloidin (Invitrogen, San Diego, CA, United States) in 0.2% (w/v) BSA-PBS (Invitrogen) to stain osteoclast cytoskeletons. Then, cells were washed using BSA-PBS (0.2% (w/v)) as well as PBS (phosphate-buffered saline), mounted using ProLong Gold anti-fade medium (Invitrogen). Observation and quantification of F-actin ring formation in each sample was done by confocal microscopy (Leica TCS SP8, Germany).
4
Immunostaining Protocols for Bladder Tissues
5
Actin Staining and SEM Imaging Protocol
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Actin staining was performed
on days 2 and 4. Therefore, the cells were washed two times with phosphate-buffered
saline (PBS, Sigma-Aldrich, Germany) and then fixated in 4% formaldehyde/PBS
(Sigma-Aldrich, Germany) at room temperature for 10 min. Then, the
samples were rinsed with PBS and the cells were permeabilized with
0.5% Triton/PBS (Sigma-Aldrich, Germany) at 4 °C for 15 min.
The specimens were then incubated in 1% bovine serum albumin (BSA)/PBS
(Sigma-Aldrich, Germany) for 5 min at 37 °C followed by the addition
of rhodamine conjugated phalloidin (1:1000 in 1% BSA/PBS, Life Technologies,
USA) and incubation for 1 h at 37 °C. Subsequently, the samples
were washed 3 times for 5 min with 0.5% Tween/PBS (Sigma-Aldrich,
Germany) followed by washing with PBS for 5 min. Next, 70 μL
Prolong gold (containing 4′,6-diamidino-2-phenylindole (DAPI),
Life Technologies, USA) were added to the cells and the samples were
mounted on glass slides and observed using a fluorescence microscope
(ZOE fluorescent cell imager, Bio-Rad, USA).
Consequently, the
stained specimens were rinsed 2 times with distilled water for 5 min.
The cells were then dehydrated in a series of graded ethanol/PBS solutions
(Sigma-Aldrich, Germany) as follows: 15 min in 50%, 20 min in 70%,
and 20 min in 96%. The specimens were allowed to dry overnight and
were gold sputtered for SEM imaging.
on days 2 and 4. Therefore, the cells were washed two times with phosphate-buffered
saline (PBS, Sigma-Aldrich, Germany) and then fixated in 4% formaldehyde/PBS
(Sigma-Aldrich, Germany) at room temperature for 10 min. Then, the
samples were rinsed with PBS and the cells were permeabilized with
0.5% Triton/PBS (Sigma-Aldrich, Germany) at 4 °C for 15 min.
The specimens were then incubated in 1% bovine serum albumin (BSA)/PBS
(Sigma-Aldrich, Germany) for 5 min at 37 °C followed by the addition
of rhodamine conjugated phalloidin (1:1000 in 1% BSA/PBS, Life Technologies,
USA) and incubation for 1 h at 37 °C. Subsequently, the samples
were washed 3 times for 5 min with 0.5% Tween/PBS (Sigma-Aldrich,
Germany) followed by washing with PBS for 5 min. Next, 70 μL
Prolong gold (containing 4′,6-diamidino-2-phenylindole (DAPI),
Life Technologies, USA) were added to the cells and the samples were
mounted on glass slides and observed using a fluorescence microscope
(ZOE fluorescent cell imager, Bio-Rad, USA).
Consequently, the
stained specimens were rinsed 2 times with distilled water for 5 min.
The cells were then dehydrated in a series of graded ethanol/PBS solutions
(Sigma-Aldrich, Germany) as follows: 15 min in 50%, 20 min in 70%,
and 20 min in 96%. The specimens were allowed to dry overnight and
were gold sputtered for SEM imaging.
6
Immunostaining for Amyloid-beta Aggregation
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For immunostaining,
fluorescence images were collected after 1 and 5 days of cell culture.
Samples were washed twice with PBS, fixed in 10% neutral buffered
formalin for 30 min at 4 °C, permeabilized with 0.1% Triton X-100
in PBS for 5 min, and blocked with 3% BSA in PBS for 30 min at room
temperature. To evaluate the accumulation of Aβo,
a primary antibody (oligomer polyclonal antibody, clone A11, rabbit,
1:200 dilution in 1% w/v BSA/PBS, Life Technologies) against Aβ
oligomeric forms or a primary antibody against Aβf aggregates (biotin anti-β-amyloid, 1–16 antibody, mouse
IgG1, 1:200 dilution in 1% w/v BSA/PBS, Biolegend) was employed, followed
by the secondary antibody rabbit anti-mouse Alexafluor-488 (1:500
dilution in 1% w/v BSA/PBS, anti-mouse, Invitrogen). A phalloidin-TRITC
conjugate was used (1:200 dilution in PBS for 30 min, Sigma) to assess
the cytoskeleton organization. Nuclei were counterstained with 1 mg/mL
of 4,6-diamidino-2-phenylindole (DAPI, Sigma) for 30 min. Samples
were washed with PBS and placed in an imaging dish for confocal microscopy
(Leica, TCS SP8).
fluorescence images were collected after 1 and 5 days of cell culture.
Samples were washed twice with PBS, fixed in 10% neutral buffered
formalin for 30 min at 4 °C, permeabilized with 0.1% Triton X-100
in PBS for 5 min, and blocked with 3% BSA in PBS for 30 min at room
temperature. To evaluate the accumulation of Aβo,
a primary antibody (oligomer polyclonal antibody, clone A11, rabbit,
1:200 dilution in 1% w/v BSA/PBS, Life Technologies) against Aβ
oligomeric forms or a primary antibody against Aβf aggregates (biotin anti-β-amyloid, 1–16 antibody, mouse
IgG1, 1:200 dilution in 1% w/v BSA/PBS, Biolegend) was employed, followed
by the secondary antibody rabbit anti-mouse Alexafluor-488 (1:500
dilution in 1% w/v BSA/PBS, anti-mouse, Invitrogen). A phalloidin-TRITC
conjugate was used (1:200 dilution in PBS for 30 min, Sigma) to assess
the cytoskeleton organization. Nuclei were counterstained with 1 mg/mL
of 4,6-diamidino-2-phenylindole (DAPI, Sigma) for 30 min. Samples
were washed with PBS and placed in an imaging dish for confocal microscopy
(Leica, TCS SP8).
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
ELISA-based Virus Binding Assay
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ELISAs were performed to examine the H1N1-virus- or HA-binding activities of sera, culture supernatant and purified antibodies. Briefly, inactivated whole H1N1, H5N1 and H7N9 virus were coated on a 96-well plate (Maxisorb, Nunc) at 4 °C overnight followed by blocking with 1% bovine serum albumin (BSA)/PBS (Invitrogen) at 37 °C for 2 h. The serial diluted samples were incubated in wells at 37 °C for 2 h. After a complete wash, the horseradish-peroxidase-labeled anti-human IgG (dilution of 1∶5000) was added and incubated at 37 °C for 1 h. 3,3′,5,5′-tetramethylbenzidine/H2O2 was added to develop color, and the reaction was stopped with 50 µl of H2SO4. The amount of chromogen produced was measured based on absorbance at 410 nm and 630 nm using an ELISA reader (SpectraMax, MD, US).
9
Visualization of Osteoclast F-actin Rings
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The osteoclasts were fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilized for 5 min with 0.1% v/v Triton X-100. The cells were then incubated with Alexa-Fluor 647 phalloidin (Invitrogen, San Diego, CA, USA) diluted in 0.2% (w/v) BSA-PBS (Invitrogen, San Diego, CA, USA) for 1 h at room temperature and washed with 0.2% w/v BSA–PBS and PBS, and DAPI was used for nuclei staining. The F-actin ring distribution was measured using the LSM5 confocal microscope (Carl Zeiss, Oberkochen, Germany). The fluorescence images were processed using the Zeiss ZEN software, and the number of intact F-actin rings was counted using Image J.
10
Quantifying Endogenous Abscisic Acid
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Endogenous ABA was extracted from the frozen samples described above. Extraction in non-oxidative methanol: water (80:20, v/v), pre-purification through SepPak C18 cartridges (Waters, Milford, MA, USA) and HPLC fractionation using a Nucleosil C18 column (Macherey-Nagel, Germany) have been described previously30 (link). The ELISA procedure was developed based upon the competition, for a limited amount of monoclonal anti-ABA antibody (1:2000 in 5% BSA/PBS; Invitrogen, CA, USA), between a standard ABA-BSA conjugate adsorbed onto the wells of a microtitration plate and free ABA extracted from the samples. Bound antibodies were labelled with a peroxidase-conjugated goat antibody raised against mouse immunoglobulins (Sigma-Aldrich, CA, USA), and peroxidase activity was then measured. A standard curve was established on each microtitration plate.
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