IF-FISH was performed as described before (Celli and de Lange 2005 (link)) with minor modifications. Paraformaldehyde-fixed cells on coverslips were permeabilized for 5 min in 0.1% Triton X-100, 20 mM HEPES-KOH (pH 7.9), 50 mM NaCl, 3 mM MgCl2, and 300 mM sucrose; washed twice with PBS for 5 min; and blocked in PBG (0.2% [w/v] cold-water fish gelatin [Sigma], 0.5% [w/v] BSA [Sigma] in PBS) for 30 min at room temperature. Cover slips were incubated with primary antibody in PBG for 2 h at room temperature, washed three times with PBS for 5 min, incubated with a fluorescently labeled secondary antibody for 1 h at room temperature, washed three times in PBS for 5 min, fixed with 3% paraformaldehyde for 5 min at room temperature, and washed three times with PBS for 5 min. A FITC-OO-[CCCTAA]3 PNA probe (Applied Biosystems) dissolved in 70% formamide, 1 mg/mL blocking reagent (Roche), and 10 mM Tris-HCl (pH 7.2) was added. After denaturation (7 min at 80°C), hybridization was for 2 h at room temperature followed by washes in 70% formamide, 10 mM Tris-HCl (pH 7.2), and PBS.
Cold water fish gelatin
Manufactured by Merck Group
Sourced in United States
Cold-water fish gelatin is a type of gelatin derived from the skin and bones of cold-water fish. It is a versatile, protein-based ingredient used in various applications within the food, pharmaceutical, and cosmetic industries.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510 meta, by Zeiss (3 mentions)
Blocking reagent, by Roche (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Fluoview 1000 confocal microscope, by Olympus (1 mentions)
Eclipse ni u fluorescent microscope, by Nikon (1 mentions)
Ds ri2 camera, by Nikon (1 mentions)
Rnase solution, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 streptavidin, by Jackson ImmunoResearch (1 mentions)
Mowiol, by Merck Group (1 mentions)
Plan apochromat 63x 1.4 oil dic objective, by Zeiss (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Graphene oxide, by Merck Group (1 mentions)
κ carrageenan, by Merck Group (1 mentions)
Tris hcl, by Merck Group (1 mentions)
Genipin, by Merck Group (1 mentions)
Collagenase from clostridium histolyticum, by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Cacl2, by Merck Group (1 mentions)
Mowiol mounting medium, by Merck Group (1 mentions)
10 protocols using cold water fish gelatin
1
Telomere IF-FISH Protocol with Modifications
2
Lens Epithelial Flat Mount Immunostaining
3
Immunofluorescent Detection of Tight Junction Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antigen retrieval was achieved by immersing deparaffinized slide sections into 1X sodium citrate buffer (10 mM, pH 6, 0.05% Tween 20; Sigma, St. Louis, MO, USA) preheated to 95–100 °C. Slides were allowed to cool on a rocker in the citrate buffer for 20 min then washed 3 times in 1X PBS for 5 min each. Sections were blocked in 2% cold-water fish gelatin (Sigma, Inc., St. Louis, MO, USA) in 0.2% Triton for 1 h, then exposed to mouse anti-claudin-5 (1:100), mouse anti-claudin-6 (1:100) or rat anti-zonula occudens-1 (1:100) in blocking buffer for 24 h at 4 °C. Slides were washed 5 times in 1X PBS with 1% Tween 20 for 5 min each then treated with anti-mouse (1:400) or anti-rat (1:500) Alexa Fluor 594 and allowed to incubate in a dark chamber for 1 h at room temperature. Slides were further washed 4 times in 1X PBS. Negative controls were performed by omitting the primary antibody. Slides were mounted in FluoroshieldTM mounting solution with DAPI counterstain (Sigma, St. Louis, MO, USA) and viewed using a Nikon Eclipse Ni-U fluorescent microscope equipped with a digital Nikon DS-Ri2 camera and NIS Elements BR software, Version 5.21.01 (Nikon Instruments Inc., Nikon, Japan). Images were captured at the same exposure time to compare changes in expression over time.
4
Telomere Length Analysis by ImmunoFISH
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ImmunoFISH was performed with some changes to the protocol45 . Human VSMCs were grown on coverslips and fixed and permeabilized with a 1:1 methanol to acetone solution. Subsequently, cells were washed, incubated with blocking buffer (2% Cold Water Fish Gelatin (Sigma) and 5% BSA in PBS), and stained with primary or isotype-control antibodies overnight at 4 °C. Cells were then washed and incubated with secondary antibody for 1 h at RT. All primary and secondary antibodies were diluted using a blocking buffer. Following secondary antibody incubation, cells were washed once with blocking buffer before being re-fixed with methanol/acetone for 2 min, treated with 0.1 mg ml−1 RNase solution (Invitrogen), and incubated for 20 min at 37 °C. After dehydration in ice-cold 70%, 85%, and 100% ethanol, coverslips were air-dried prior to hybridization with a Cy3-TelC PNA probe, following the manufacturer’s instructions (PNA Bio inc). Next, cells were then washed with PBS, incubated with DAPI, and mounted for imaging with confocal microscopy.
5
Immunofluorescence Staining of U2OS Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
U2OS cells were grown on glass coverslips and fixed in 4% paraformaldehyde/PBS for 10 minutes at room temperature, permeabilized for 5 minutes in PBS containing 0.5% Triton X-100 and blocked by incubation in IF blocking buffer (PBS containing 0.5% cold water fish gelatin (Sigma, Vienna, Austria)) for 15 minutes. All primary and secondary antibody dilutions were prepared in IF blocking buffer and applied for 60 minutes. DNA was stained with 1 µg/ml DAPI (Sigma, Vienna, Austria) and coverslips were mounted on microscopy slides in Mowiol (Sigma, Vienna, Austria). All samples were imaged using a confocal laser scanning microscope (LSM-Meta 510, Carl Zeiss AG, Oberkochen, Germany) using a Plan-Apochromat 63x/1.4 Oil DIC objective. Primary antibodies used for immunofluorescence were the same as used for Western Blotting. In addition, an Alexa Fluor™ 647-Streptavidin (016-600-084, Jackson ImmunoResearch, West Grove, Pennsylvania, USA) was used for detection of biotinylated proteins. Secondary antibodies conjugated to Alexa-488 were purchased from Molecular Probes and secondary antibodies conjugated to Cy5 or TexasRed from Jackson ImmunoResearch (West Grove, Pennsylvania, USA).
6
Gelatin-Carrageenan Composite with Graphene Oxide
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cold water fish gelatin, κ-carrageenan, genipin (HPLC grade > 98%), and graphene oxide (powder, 15–20 sheets, 4–10% edge oxidized) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Additionally, the reagents involved in the enzyme degradation study, collagenase from Clostridium histolyticum, Tris-HCl, NaN3, CaCl2, and EDTA were purchased from Sigma-Aldrich (St. Louis, MO, USA).
7
Histological Evaluation of Muscle Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For histological
evaluation, samples were washed in 0.1 M PBS, pH
7.4, fixed in 4% formaldehyde in 0.1 M PBS, pH 7.4, dehydrated through
graded ethanol series, cleared in xylene, embedded in paraffin, and
cut into 5 μm thick sections. Sections were stained with hematoxylin
and eosin (H&E) or toluidine blue (2.5% in 5% Borax solution)
and by immunofluorescence using an antibody against myosin.17 (link) Briefly, sections were incubated in 0.1 M citrate
buffer for 60 min, blocked by 0.15% glycine in 0.1 M PBS, pH 7.4,
for 30 min, and in 1% (w/v) bovine serum albumin (BSA), 5% (v/v) normal
goat serum, 0.1% (w/v) cold water fish gelatin (Sigma-Aldrich), and
0.1% (v/v) Triton X-100 in 0.1 M PBS, pH 7.4, for another 30 min.
Sections were incubated with a primary antibody against myosin (1:400,
MY-32, Sigma-Aldrich) for 60 min, followed by a secondary antibody
goat antimouse IgG (H + L) Alexa 488 for 60 min and 10 μg/mL
4′,6-diamidino-2-phenylindole (DAPI; Roche Diagnostics GmbH,
Mannheim, Germany) to stain nuclei. Control sections were incubated
with the secondary antibody only. After each step, sections were washed
with PBS. Sections were enclosed in Mowiol mounting medium (Sigma-Aldrich).
evaluation, samples were washed in 0.1 M PBS, pH
7.4, fixed in 4% formaldehyde in 0.1 M PBS, pH 7.4, dehydrated through
graded ethanol series, cleared in xylene, embedded in paraffin, and
cut into 5 μm thick sections. Sections were stained with hematoxylin
and eosin (H&E) or toluidine blue (2.5% in 5% Borax solution)
and by immunofluorescence using an antibody against myosin.17 (link) Briefly, sections were incubated in 0.1 M citrate
buffer for 60 min, blocked by 0.15% glycine in 0.1 M PBS, pH 7.4,
for 30 min, and in 1% (w/v) bovine serum albumin (BSA), 5% (v/v) normal
goat serum, 0.1% (w/v) cold water fish gelatin (Sigma-Aldrich), and
0.1% (v/v) Triton X-100 in 0.1 M PBS, pH 7.4, for another 30 min.
Sections were incubated with a primary antibody against myosin (1:400,
MY-32, Sigma-Aldrich) for 60 min, followed by a secondary antibody
goat antimouse IgG (H + L) Alexa 488 for 60 min and 10 μg/mL
4′,6-diamidino-2-phenylindole (DAPI; Roche Diagnostics GmbH,
Mannheim, Germany) to stain nuclei. Control sections were incubated
with the secondary antibody only. After each step, sections were washed
with PBS. Sections were enclosed in Mowiol mounting medium (Sigma-Aldrich).
8
Cirish Root Powder Extraction and Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For this research, the Cirish root powder was purchased from the local medical plant market, Mashhad, Iran and then were passed through a 60‐m sieve screen and stored at 4°C for further use. Cold‐water fish gelatin was purchased from Sigma‐Aldrich.
9
Immunofluorescence Imaging of Neutrophils
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Neutrophils were fixed on uncoated glass coverslips with 2% paraformaldehyde (PFA), permeabilized for 5 minutes with 0.5% Triton X-100, and then blocked at room temperature for 30 minutes in 1% bovine serum albumin, 5% normal donkey serum, and 3% cold-water fish gelatin (Sigma-Aldrich). To promote attachment of unstimulated cells, neutrophils were first plated for 5 minutes in RPMI without human serum albumin, after which culture medium was added (RPMI with 0.05% HSA). Primary antibodies used were: anti-Ki67 (Abcam SP6), anti-a/b Tubulin (Cell Signaling Technology [CST]), anti-pericentrin (Abcam), anti-Lamin A/C phospho-ser392 (Biorbyt), anti-Cdk6 (Santa Cruz, C-21), anti-elastase (EMD), anti-g tubulin (Thermo) and anti-chromatin (reacts with a complex of Histone 2A, Histone 2B and DNA, (Losman et al., 1992) ). Samples were stained with primary antibodies for 1h at 37 C in blocking buffer, followed by secondary antibodies conjugated to Alexa Fluor 488 or 568 (Invitrogen) together with Hoechst 33342. Coverslips were mounted in Mowiol and images were taken with a Leica SP8 confocal microscope.
10
Immunofluorescence Staining of OR2J3 in QGP-1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The QGP-1 cells were cultured on 30-mm coverslips until they were 80% confluent. After a washing step using PBS, the cells were fixed with ice-cold acetone for 5 min. To avoid nonspecific antibody binding, the cells were blocked in 1% cold-water fish gelatin (Sigma-Aldrich) diluted in TBS with 0.05% Triton X-100 (Sigma-Aldrich), followed by incubation with the primary antibody. A primary antibody directed against OR2J3 (rabbit polyclonal) (SAB4501918, Sigma-Aldrich) was used. The cells were co-incubated with 4′-6-diamidin-2-phenylindol (DAPI) fluorescent dye to label the nuclei. The fluorophorecoupled secondary antibodies Alexa Fluor 488 nm (goat anti-rabbit) (#A-11034, Thermo Fisher Scientific) and 546 nm (goat anti-rabbit) (#A-11010, Thermo Fisher Scientific) were used, and cells were coated with ProLong Antifade Gold (Thermo Fisher Scientific). The fluorescent signals were detected using confocal microscopy (Zeiss LSM 510 Meta, Oberkochen, Germany) with a 40× oil immersion objective and the Leica Application Suite software (LAS, Leica). The images were processed using Corel Draw X5 (Corel, Ottawa, ON, Canada).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!