Apoptosis immunofluorescence staining was performed using the In situ Cell Death Detection Kit, TMR Red (Roche Applied Science, Mannheim, Germany). Initially, 10 µg/ml of proteinase K (Roche Applied Science, Mannheim, Germany) in 10 mmol/L Tris/HCl (pH 7.4) was applied to the MG specimens and left for 15 minutes at room temperature. After washing the samples with 0.1M PBS twice, TUNEL reaction mixture (Roche Applied Science, Mannheim, Germany) was added on to the samples and the label solution on the negative control samples, then incubated at 37°C for 60 minutes in a dark room. The specimens were rinsed 3 times in 1M PBS for 5 minutes each, and then 100 µl of 0.5 µg/ml DAPI diluted in Tris-buffered saline and Tween-20 were added to the samples for 5 minutes at room temperature. Finally, the specimens were washed with 1M PBS and mounted with aqueous mounting medium Permafluor (Beckman Coulter, Marseille, France). Sections were examined and photographed with an epifluorescence microscope (Axioplan2 Imaging, Carl Zeiss, Jena, Germany).
Permafluor
Manufactured by Beckman Coulter
Sourced in United States, Germany
Permafluor is a liquid scintillation cocktail used in radioactive liquid scintillation counting. It is designed to efficiently detect and measure radioactive samples.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer z1 bio, by Zeiss (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Axioplan 2 imaging, by Zeiss (1 mentions)
Proteinase k, by Roche (1 mentions)
Tunel reaction mixture, by Roche (1 mentions)
Lsm700 2ch urgb, by Zeiss (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Alexa fluor 568 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Anti gsdmd, by Cell Signaling Technology (1 mentions)
Ab21027, by Abcam (1 mentions)
Axioplan 2 microscope, by Zeiss (1 mentions)
Axiocam, by Zeiss (1 mentions)
Dm irbe, by Leica (1 mentions)
Tcs sp, by Leica (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Vector trueview quenching kit, by Vector Laboratories (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
780 confocal microscope, by Zeiss (1 mentions)
Rabbit anti gfp antibody, by Thermo Fisher Scientific (1 mentions)
Cy3 conjugated donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
11 protocols using permafluor
1
Apoptosis Immunofluorescence Staining Protocol
2
Immunofluorescence Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were grown on 13 mm round glass coverslips. After treatment as described, cells were placed on ice, washed twice with PBS and fixed with 0.37% PFA in phosphate-buffered saline (PBS) for 10min. Cells were then washed twice in PBS and treated by cold blocking buffer for 1 h. After sequential treatment with NH4Cl (50 mM in 20 mM glycine) for 10 min, the indicated antibody (1:200 in bovine serum albumin) was added and incubated overnight at 4°C. After an additional incubation for 1 h at room temperature with Hoechst 33258 and fluorescein isothiocyanate-conjugated secondary antibody (Invitrogen) (1:400 in bovine serum albumin), the slides were mounted in anti-fading solution (Permafluor, Beckman Coulter, Krefeld, Germany) and stored at 4°C, followed by confocal laser-scanning microscopy.
3
Assessing AQP4 Antibody Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
AQP4 live transfected human embryonic kidney cells (HEK293) expressing the untagged AQP4-M23 isoform were incubated in 4x diluted serum for 1 h, washed and incubated in fluorescein-conjugated goat anti-human IgG (MP Biomedicals, Aurora, OH, USA) for 30 min. Cells were fixed in 95% ethanol and mounted in the prolong antifade mounted media Permafluor (Beckman Coulter, Fullerton, Ca, USA). Image were captured by confocal microscopy Fluoview (Olympus Tokyo). Ab-seropositivity was based on comparison with mock-transfected cells that did not express AQP4.
4
Immunofluorescence Analysis of XBP1s in HuH-7 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following treatment with GGA or other compounds, HuH-7 cells grown on glass inserts in a 24-well plate were rinsed with Ca-free PBS (PBS(-); Sigma Aldrich) and fixed for 40 min with 4% paraformaldehyde containing 2% sucrose in PBS(-), and then rinsed with PBS(-). Cells were then permeabilized with 0.5% Triton X-100 and nonspecific binding blocked with 10% FBS. Next, cells were incubated at 4°C overnight with polyclonal anti-XBP1s antibody, followed by 2.5 h incubation with Alexa-488-labeled goat anti-rabbit IgG antibody (Invitrogen, Molecular Probes, Tokyo, Japan). After rinsing with PBS(-), cells were mounted in PermaFluor (Beckman Coulter, Brea, CA, USA), covered on a glass slide, and observed under a confocal laser-scanning fluorescence microscope, LSM700 2Ch URGB equipped with Axio Observer Z1 Bio (Carl Zeiss, Göttingen, Germany).
5
Immunofluorescence Staining of GSDMD and NF-κB
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence staining was performed using cultured HuH-7 cells grown on glass inserts in a 24-well plate. Briefly, after fixation, the cells were incubated at 4°C overnight with anti-GSDMD (Cell Signaling Technology) or anti-nuclear factor-κB (NF-κB) p65 (D14E12, Cell Signaling Technology) antibodies, followed by 1 h of incubation with Alexa 488-labelled goat anti-rabbit IgG (Invitrogen, Molecular Probes, Tokyo, Japan) or Alexa Fluor 568–conjugated goat anti-mouse IgG (Invitrogen). After rinsing with PBS (−), the cells were mounted in PermaFluor (Beckman Coulter, La Brea, CA, U.S.A.) containing DAPI on a glass slide and observed under a confocal laser-scanning fluorescence microscope (LSM700 2Ch URGB with Axio Observer Z1 Bio; Carl Zeiss, Göttingen, Germany).
6
Immunofluorescence Assay of Aquaporin Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence assay was performed as described previously. Briefly, frozen sections of lacrimal glands and whole eye tissues (5 μm thick) were air-dried, fixed in cold acetone for 10 minutes, and blocked with 5% donkey serum in phosphate-buffered saline (PBS) for 30 min. Sections were then incubated overnight at 4°C with the primary antibodies as follows: rabbit anti-AQP3 polyclonal antibody (sc-20811, H-80, Santa Cruz Biotechnology, Inc., Santa Cruz, CA); rabbit anti-AQP5 polyclonal antibody (sc-28628, H-200, Santa Cruz); rabbit anti-AQP8 (sc-28624, H-85, Santa Cruz); rabbit anti-AQP9 (ab105148, Abcam, Cambridge, MA); and goat anti-α-SMA polyclonal antibody (ab21027, Abcam). The sections were next incubated for 30 min with the species-appropriate secondary antibodies as follows: AlexaFlour® 488-conjugated donkey anti-goat IgG (Life Technologies, Carlsbad, CA) and AlexaFlour® 555-conjugated anti-rabbit goat IgG (Life Technologies) and then washed with PBS and mounted using Permafluor (Beckman Coulter Inc., Miami, FL). Images were acquired using an Axioplan 2 microscope (Carl Zeiss Inc., Thornwood, NY) equipped with a digital camera (Axiocam; Carl Zeiss Inc.).
7
Immunocytochemical Analysis of Muscle Differentiation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were grown on X-well slides (Sarstedt, Nümbrecht, Germany) coated with GelTrex and differentiated as described above. After 5 days of differentiation, they were washed with PBS, fixed in PBS containing 4% formaldehyde (pH 7.4) for 20 min, quenched with 150 mM glycine in PBS for 10 min and permeabilised with 0.1% Triton X100 for 2 min. Blocking was performed with 1% NGS, 0.05% Tween 20 in PBS for 30 min. Antibodies recognizing CD56, MYH1 + 2, or MYH7 (Table 1a ) were diluted in blocking solution and added for 1 h at room temperature. After washing thrice in PBS, the second antibody (Table 1b ), diluted in blocking solution, was added for 2 h. After another wash, nuclei were stained using TO-PRO3 (Invitrogen, Carlsbad, California, USA) before mounting in PermaFluor (Beckman Coulter, Krefeld, Germany). Images were obtained using a DM-IRBE equipped with a TCS-SP and a DCF340FX (Leica, Wetzlar, Germany).
8
BiFC Assay on Chick Embryos
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the BiFC assay on chick embryos, we used previously described vectors containing the N- and C-terminal halves of Venus (amino acids 1–172 and 173–239; Strobl-Mazzulla et al., 2010 (link)). Full-length JmjD2A, 14-3-3, and SAP18 (used as a MOCK protein) were cloned in-frame upstream of the Venus halves–containing vector. Chick embryos were electroporated with the vectors at stage 5 and then cultured in 0.5 ml of albumen in tissue culture dishes for 16 h. Embryos were fixed in 4% PFA for 20 min at room temperature, washed in PBS, and visualized as a whole mount. Some embryos were subsequently embedded in gelatin, cryostat sectioned at 16 μm, cover slipped using Permafluor (Beckman Coulter), and photographed.
9
Immunofluorescence Staining of Sheep Explants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Prior to immunofluorescence staining, 5 µm thick paraffin sections of sheep explants were dewaxed and rehydrated to water. Antigen retrieval was carried out by microwaving the slides in 0.1 M citrate buffer. To reduce non-specific binding, the slides were incubated with 10% normal goat serum in 2% bovine serum albumin (BSA -Sigma) for 40 min. Specimens were then incubated overnight in a moist chamber with a cocktail of antibodies consisting smooth muscle alpha actin (mouse from DAKO) and CD31 (rabbit- Abcam) Negative controls consisted of 10% normal goat serum in 2% BSA in PBS. After thorough washing, all the specimens were incubated with secondary antibodies, goat anti rabbit Alexa Fluor 634 for rabbit polyclonals and goat anti mouse Alexa Fluor 488 (Life Technologies) for 1 h at 1:1000 dilution. After washing several times with PBS sections were incubated with DAPI (1:5000) (sigma) for 15 min and to reduce autofluorecence, sections were incubated with Vector TrueView quenching kit (Vector laboratories) for 3 min and mounted using Permafluor (Beckman Coulter). Stained sections were imaged using a Zeiss LSM 880 confocal microscope.
10
Melanopsin Immunostaining in Mouse Retina
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!