For immunostaining of RAP1, TRF1, TRF2 together with lamin B1, cells grown on coverslips, 24 or 48 h after transfection, were pretreated with extraction buffer (50 mM Tris–HCl, 150 mM NaCl, 1% Trition-X100, 1 mM NaF, 1 mM Na3VO4, 1 mM PMSF, as described in (64 (link)), 2 min on ice before fixation in 2% PFA (10 min)). For single staining with lamin B1, cells were directly fixed in 4% PFA or in ice-cold 100% methanol (10 min). Then, cells were saturated in PBS with 2% BSA-0.05% Tween and stained for 1 h with primary antibodies (mouse anti-TRF1 (Sigma T1348), mouse anti-RAP1 (ab14404, Abcam), mouse anti-TRF2 (Imgenex 124A or Santa Cruz B5) and rabbit anti-lamin B1(ab16048, Abcam), then washed and incubated 1 h with the secondary antibodies (alexa fluor-488 (Life Technologies) or mouse primary antibody and alexa fluor -594 (Life Technologies) for rabbit primary antibody). Nuclei were then counterstained with DAPI and slides were mounted with fluoromount mounting medium (Southern Biotech). Images were acquired on epifluorescence microscope leica DM5500B using a 63×-oil objective and analyzed with ImageJ software.
Fluoromount mounting medium
Manufactured by Southern Biotech
Sourced in United States, Germany
Fluoromount mounting medium is a liquid mounting solution used for preparing microscope slides. It is designed to preserve and protect fluorescent samples for long-term storage and observation.
Automatically generated - may contain errors
Lab products found in correlation
Draq5, by Thermo Fisher Scientific (2 mentions)
Leica tcs spe 2 laser scanning confocal microscope, by Leica camera (2 mentions)
Eclipse fluorescent microscope, by Nikon (2 mentions)
4 6 diamidino 2 phenylindol dapi, by Roche (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Ab14404, by Abcam (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Imgenex 124a, by Novus Biologicals (1 mentions)
Ab16048, by Abcam (1 mentions)
Dnase 1 solution, by Qiagen (1 mentions)
Deadend fluorometric tunel system, by Promega (1 mentions)
Confocal laser scanning microscopy, by Leica camera (1 mentions)
Phalloidin 488, by Santa Cruz Biotechnology (1 mentions)
Axiovision 4, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by AppliChem (1 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Imager d1 microscope, by Zeiss (1 mentions)
Triton x 100, by Carl Roth (1 mentions)
Axiocam mrm camera, by Zeiss (1 mentions)
12 protocols using fluoromount mounting medium
1
Immunostaining for Telomere-associated Proteins
2
Intracellular Trafficking of Polyplexes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
H1299 cells were seeded at the density of 50,000 cells/well in 24-well plates with a 12 mm circle cover glass (Fisherbrand, 12CIR-1) in each well and were incubated for 24 h. To determine intracellular distribution of polyplexes, fluorescently labeled polymer bPEI-FITC and HAI-SMCC-bPEI-FITC were formulated with 50 pmol of siRNA-Ty563 at N/P = 5. Cells were transfected with polyplexes at a siRNA concentration of 125 nM for the first 4 h and 50 nM for an additional 44 h. Cells were washed with PBS and fixed with 4% paraformaldehyde (PFA) solution in PBS (Affymetrix, Thermo Fisher) for 20 min at RT at different transfection time points (1, 4, 24, 48 h). To investigate the co-localization of the HAI peptide binding part with TfR, cells were incubated with 2 µg/mL of Tf-Texas Red and polyplexes formulated with bPEI-FITC or HAI—SMCC-bPEI-FITC and 50 pmol of siRNA at N/P = 5 for 1 h or 4 h. Slides were washed with PBS and fixed with 4% PFA. After fixation, the nuclei were stained with 5 µM of DRAQ5 (Invitrogen, Thermo Fisher) for 5 min and rinsed with PBS. Slides were mounted with a Fluoromount mounting medium (Southern Biotech, Birmingham, AL, USA). Slides were imaged by a Leica TCS SPE-II laser scanning confocal microscope (Leica, Wetzlar, Germany), and the images were exported from the Leica Image Analysis Suite (Leica).
3
Testicular Apoptosis Evaluation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TUNEL assay was performed on paraffin-embedded testis sections using the DeadEnd Fluorometric TUNEL System according to manufacturer’s instructions (Promega, Madison, WI, USA). The positive control sections were pre-incubated with DNase I solution (Qiagen, Valencia, CA, USA), while negative control sections were incubated in incubation buffer without recombinant terminal deoxynucleotidyl transferase (rTdT enzyme). Slides were mounted with fluoromount mounting medium containing DAPI (SouthernBiotech, Birmingham, AL, USA) and visualized using a Nikon Eclipse fluorescent microscope (Nikon Corporation). 10 pictures were taken for each animal and TUNEL-positive cells were counted using FIJI software (Schindelin et al., 2012 (link)).
4
Confocal Microscopy Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein expression profile was assessed using confocal laser scanning microscopy. Seeded glass cover slips and ACL tissue paraffin sections, fixed in 4% PFA, were washed with Tris buffered saline (TBS: 0.05 M Tris, 0.015 M NaCl, pH 7.6), before being incubated with protease-free donkey serum (5% diluted in TBS with 0.1% Triton X100) for cell permeabilization for 20 min at RT. Subsequently, samples were incubated with primary antibodies (listed in Table 1 ) overnight at 4 °C in a humidified chamber. Samples were rinsed with TBS prior to incubation with donkey-anti-goat or anti-rabbit-Alexa-488 (Invitrogen, CA, USA) or donkey-anti-mouse or goat-Cy3 (Invitrogen, Carlsbad, USA) coupled secondary antibodies (diluted 1:200 in TBS with 0.1% Triton ×100 and 5% donkey serum), respectively, for 1 h at RT in a humidified chamber. Cell nuclei were counterstained using 4′,6′-diamidino-2-phenylindol (DAPI, Roche, Mannheim, Germany) and phalloidin-488 (1:100, Santa Cruz Biotechnologies) to depict F-actin cytoskeletal actin architecture. Labelled cells were washed several times with TBS, before mounting with Fluoromount mounting medium (Southern Biotech, Biozol Diagnostica, Eching, Germany) and examined by using confocal laser scanning microscopy (Leica).
5
Immunofluorescence Staining of IER2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells grown on glass coverslips were washed with PBS, fixed with 100% ice-cold methanol (AppliChem, Darmstadt, Germany) for 10 min at −20 °C, then permeabilized using 0.1% Triton X-100 (Carl Roth, Karlsruhe, Germany) for 10 min at room temperature (RT). After washing with PBS, cells were incubated in 10% FBS/PBS for 30 min to block unspecific binding. Cells were incubated with IER2 antibody (ARP34401_P050; Aviva Systems Biology, San Diego, CA, USA) diluted 1:500 in blocking solution for 3 h at RT, then extensively washed with PBS. Incubation with the goat anti-rabbit AlexaFluor 546 (Thermo Fisher Scientific, Waltham, MA, USA) secondary antibody (1:1000) was performed for 1 h at RT. Cell nuclei were counterstained with 1 μg/ml DAPI (AppliChem, Darmstadt, Germany) for 5 min at RT, and coverslips were mounted in Fluoromount mounting medium (SouthernBiotech, Birmingham, AL, USA). Fluorescent signals were captured using a Zeiss Axio Imager D1 microscope equipped with an AxioCam MRm camera and AxioVision 4.7 software (Carl Zeiss, Oberkochen, Germany).
6
Immunofluorescence Staining Protocol for SV40 T Antigen
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were cultured in the same way as for the other immunofluorescence stains. Then, the cells were washed three times with TBS before being incubated with protease-free donkey serum blocking and permeabilization buffer (5% in TBS with 0.1% Triton X-100) for 20 min at RT. Subsequently, cover slips were incubated with the primary antibody (SV40 T antigen mouse anti-human, Merck-Millipore, Darmstadt, Germany, 1:50) overnight at 4 °C in a humidifier chamber. Samples were rinsed three times with TBS prior to incubation with donkey anti-mouse cyanine (Cy)3 secondary antibody (Invitrogen, Waltham, MA, USA) combined with Alexa Fluor 488 phalloidin (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) (diluted 1:200 and 1:100 in TBS with 0.1% Triton ×100 and 5% donkey serum) to visualize cytoskeletal F-actin organization for 1 h at RT in a humidifier chamber. During this incubation step, cell nuclei were counterstained using 4′,6-diamidino-2′-phenylindol (DAPI, Roche, Mannheim, Germany). Labelled cells were washed three times with TBS before being covered with Fluoromount mounting medium (Southern Biotech, Biozol Diagnostica, Eching, Germany). Photos were taken using confocal laser scanning microscopy (TCS SPEII; Leica, Wetzlar, Germany). The utilized antibodies are indicated in Table 2 .
7
TAAR-1 Immunostaining of Brain Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fixed brain tissues were processed as previously described100 . Citrate buffer antigen retrieval (ThermoFisher, Waltham, MA, USA) was applied to all tissue sections. The sections were incubated overnight at 4 °C with an anti-TAAR-1 antibody (sc398096, Santa Cruz Biotechnology) diluted 1:100 in the blocking buffer. The sections were then incubated for 2.5 h at room temperature with Alexa Fluor-488 conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA). DRAQ5 (Invitrogen) was used to stain nuclei. The sections were then mounted using Fluoromount mounting medium (Southern Biotech, Birmingham, AL, USA). The immunostaining on each slice (3 sections per slice) was imaged using the Leica TCS SPE-II laser scanning confocal microscope (Leica, Wetzlar, Germany) and averaged per rat.
8
Visualization of cellular structures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells seeded on poly-L-lysine coverslips (BD Biosciences) were fixed with 10% neutral buffered formalin solution (Sigma) for 15 min and extracted with PBS containing 0.5% Triton X-100 (Sigma) for 10 min at room temperature. After blocking with 5% BSA (Sigma), samples were incubated with indicated primary antibodies overnight at 4 °C. Samples were then washed and incubated with secondary antibodies plus DAPI (Thermo Scientific) for 1 h at room temperature. Samples were mounted onto glass slides with Fluoromount mounting medium (SouthernBiotech) and visualized by FV1200 (Olympus) confocal microscope. For live cell microscopy, cells seeded on 35 mm glass-bottom dishes (In Vitro Scientific) were stained with SYTO™ RNA select™ green fluorescence (ThermoFisher) for 20 min at 37 °C, washed and followed with 50 μM monodansylcadaverine (Sigma) plus Hoechst33342 (Invitrogen) for 30 min at 37 °C. After wash with PBS, the stained cells in FluoroBrite DMEM (ThermoFisher) were analysed with FV1200 (Olympus) confocal microscope.
9
cFos Immunohistochemistry in Mouse Brain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For cFos immunohistochemistry (IHC), mice were deeply anesthetized with pentobarbital (150 mg/kg intraperitoneally, Streuli Pharma, Switzerland) and perfused trans-cardially (4.0% paraformaldehyde, 1X PBS, pH 7.4). Brains were removed, post-fixed (4% PFA overnight), and cryoprotected (30% sucrose, 1X PBS, 4 °C, 48 h). They were then frozen and 40 μm coronal sections were cut with a sliding cryostat (Leica Microsystems, Germany).
Subsequently, free floating sections were incubated in blocking solution (1% BSA, 1X PBS, 0.3% TrytonX100) at room temperature for 1 h, followed by incubation with rabbit anti-cFos antibody (1:5000, Synaptic System, Germany, #226 003) in blocking buffer (1% BSA, 1X PBS, 0.1% TrytonX100) overnight at 4 °C under constant shaking. Sections were washed extensively with PBS Tryton 0.1% and then exposed to the secondary antibody (Alexa Fluor 647-conjugated donkey anti-rabbit IgG, Life Technologies, USA) in blocking buffer at room temperature for 2 h. After extensive washing, the sections were incubated with Hoechst (Life Technologies, USA) at 1:1000 in PBS at room temperature for 5 min. Slices were washed extensively with PBS and mounted on superfrost glass slides (ThermoScientific, USA) with Fluoromount mounting medium (SouthernBiotech, USA). Images were acquired on a virtual slide microscope (VS120, Olympus, Japan) with a 10 × objective.
Subsequently, free floating sections were incubated in blocking solution (1% BSA, 1X PBS, 0.3% TrytonX100) at room temperature for 1 h, followed by incubation with rabbit anti-cFos antibody (1:5000, Synaptic System, Germany, #226 003) in blocking buffer (1% BSA, 1X PBS, 0.1% TrytonX100) overnight at 4 °C under constant shaking. Sections were washed extensively with PBS Tryton 0.1% and then exposed to the secondary antibody (Alexa Fluor 647-conjugated donkey anti-rabbit IgG, Life Technologies, USA) in blocking buffer at room temperature for 2 h. After extensive washing, the sections were incubated with Hoechst (Life Technologies, USA) at 1:1000 in PBS at room temperature for 5 min. Slices were washed extensively with PBS and mounted on superfrost glass slides (ThermoScientific, USA) with Fluoromount mounting medium (SouthernBiotech, USA). Images were acquired on a virtual slide microscope (VS120, Olympus, Japan) with a 10 × objective.
10
Immunofluorescence Staining of Myofibres
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Myofibres were fixed in 4% PFA/PBS for 15 min, and washed 3× in PBS. Fibres were permeabilised in 0.1% triton-X/PBS for 10 min, washed 3× and blocked in 10% normal goat serum/PBS for 1 h. Fibres were then treated with primary antibodies in blocking solution overnight (β-tubulin) or for 3 h (lamin A, nesprin-1, pericentrin) at 4 °C. Fibres were washed 3× in PBS for a total of 30 min, and then treated with Alexa 594 or 488-conjugated secondary antibodies and DAPI (all at 1:1000 in PBS) for 3 h. Fibres were washed 3× in PBS for a total of 30 min and mounted in Fluoromount mounting medium (Southern Biotech) with coverslip (thickness #1.5).
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!