For visualization of actin filaments, cells were fixed with 4% electron microscopy grade paraformaldehyde (Electron Microscopy Sciences) for 10 min at room temperature (RT) and then permeabilized for 3 min with 1% Triton X. Cells were then washed three times with PBS and actin filaments were stained with Alexa Fluor 488 phalloidin or Alexa Fluor 568 phalloidin (diluted 1:100, Life Technologies) for 30 min at RT in immunofluorescence staining buffer. For visualization of mitochondria, cells were fixed with 4% electron microscopy grade paraformaldehyde (Electron Microscopy Sciences) for 10 min at RT and then permeabilized for 5 min with 0.1% Tween 20. Cells were washed three times with PBS and incubated with an antibody to TOM-20 (rabbit, 1:500, CST) at 4°C overnight. Cells were then washed twice in PBS and incubated with (goat anti-rabbit Alexa Fluor 568, 1:1,000) for 2 h and then washed three times in PBS before mounting with Prolong Diamond (Life Technologies).
Electron microscopy grade paraformaldehyde
Manufactured by Electron Microscopy Sciences
Sourced in United States
4% electron microscopy grade paraformaldehyde is a fixative solution used in the preparation of samples for electron microscopy. It is a 4% concentration of paraformaldehyde, a polymer of formaldehyde, in an aqueous solution. This product is specifically formulated and purified for use in electron microscopy applications.
Automatically generated - may contain errors
Lab products found in correlation
Prolong diamond, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 568 phalloidin, by Thermo Fisher Scientific (2 mentions)
Lsr 2, by BD (1 mentions)
Rnasin plus rnase inhibitor, by Promega (1 mentions)
Recoverall total nucleic acid isolation kit, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Rabbit anti tom20, by Proteintech (1 mentions)
Mouse anti dlp1, by BD (1 mentions)
Prolong gold antifade mounting reagent, by Thermo Fisher Scientific (1 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Merck Group (1 mentions)
Ix81 spinning disk confocal microscope, by Olympus (1 mentions)
Volocity 6, by PerkinElmer (1 mentions)
6 protocols using electron microscopy grade paraformaldehyde
1
Visualizing Actin Filaments and Mitochondria
2
Mesothelin Surface Expression Analysis
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Panc02 cells were washed with DPBS and cells harvested by Trypsin-free Cell Dissociation Solution (Sigma-Aldrich). Cells were incubated in cold flow buffer (DPBS with 0.2 g % BSA + 0.01 % sodium azide) with 1:1000 of MBL B35 rat monoclonal antibody or normal rat IgG for background control. Following 30 minute incubation on ice, donkey anti-rat Cy5 conjugated secondary antibody was added at 1:100. Cells were incubated for an additional 30 minutes on ice, washes repeated and cells were fixed with 1 % electron microscopy grade paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) diluted in DPBS. Data were acquired on a BD LSR II (BD Biosciences) and data analysis was conducted using BD FACSDiva or FlowJo software. We found that trypsin cleaves mesothelin from the surface, so in experiments with adherent cells, cells were dissociated from the surface using trypsin-free solutions.
3
RNA Isolation from Sorted Cells
4
Immunofluorescence Staining of Actin Cytoskeleton
5
Immunofluorescence Staining of Mitochondrial Proteins
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Cells were seeded onto coverslips and cultured for 2 hr prior to fixing and staining. Thereafter cells were fixed with 4% electron microscopy grade paraformaldehyde (Electron Microscopy Sciences) for 10 min at RT and then permeabilized for 3 minutes with 0.2% Triton-X 100 (Millipore Sigma). Over-extracted cells were treated with 0.4% Triton-X 100 for 10 min. Cells were then washed three times with DPBS and stained overnight at 4°C with primary antibodies diluted in PBS. Next, they were washed twice with PBS for 5 min, incubated with secondary antibodies (diluted 1:1000) for 2 hr at RT in PBS. Actin filaments were stained with Alexa Fluor 488, 568 or 647 phalloidin (Life Technologies) for 30 min at RT in DPBS. Cells were washed three times with DPBS before mounting with Prolong Diamond (Life Technologies). The following antibodies were used: Rabbit anti-Tom 20 (cat# 11802-1-AP, Proteintech) 1:300 dilution; rabbit-anti-parkin (cat # PA5-13399, Invitrogen) 1:50 dilution, mouse anti-DLP1 (cat# 611113 Clone 8/DLP1 (RUO), BD biosciences) 1:100 dilution, Preabsorbed secondary antibodies used were anti-mouse IgG 647, anti-rabbit IgG 568 at a 1:1000 dilution. Coverslips were mounted onto slides using Prolong Diamond (Thermo Fisher). All slides were cured at RT in the dark for 2 days prior to imaging.
6
Immunofluorescence Staining of A549 Cells
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A549 cells on coverslips were fixed with 4% electron microscopy-grade paraformaldehyde (Electron Microscopy Sciences; Hatfield, PA, USA) in PBS for 10 min at room temperature and permeabilized with 0.5% Triton X-100 in PBS for 10 min. Coverslips were then washed three times with PBS, after which they were incubated in blocking buffer for 30 min at room temperature. Coverslips were then incubated with primary antibodies diluted in blocking buffer for 45 min at room temperature. Coverslips were washed three times with PBS, and then they were incubated with secondary antibodies (1:1000; Invitrogen) and DAPI (4′,6-diamidino-2-phenylindole; 1 μg/mL; Sigma Aldrich) in blocking buffer for 45 min at room temperature. Samples were then washed three times with PBS and once with de-ionized water; then, they were mounted onto microscope slides with Prolong Gold anti-fade mounting reagent (Life Technologies). Samples were imaged using an Olympus IX-81 spinning-disk confocal microscope equipped with a 60× PlanApo N oil objective. Images were analyzed using Volocity 6.2.1 software (PerkinElmer; Waltham, MA, USA).
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