Cells were fixed with 10% neutral buffered Formalin (Sigma; HT501128) for 20 min at room temperature and permeabilized for 10 min in 0.2% Triton X-100 (Sigma; 93418) in PBS. Samples were then incubated with an anti-vinculin antibody (Sigma; V9131; 1:400). Next, samples were washed several times in PBS and further incubated with an anti-mouse antibody (Thermo; A21202) together with fluorescent phalloidin (Thermo; A22284) and NucBlue (Invitrogen; R37605) for 1 h. Finally, samples were washed with PBS and mounted in Mowiol. Confocal images were acquired using an inverted spinning-disk confocal microscope (iMic; FEI Photonics) using a 60× objective (NA 1.35). For morphological analysis in the free-floating state, cells were trypsinized for 5 min, centrifuged at 250 × g, and resuspended in PBS. F-actin and nuclear staining were performed using the same protocol as in the case of adhering cells. In both conditions, images were preprocessed with ImageJ (National Institutes of Health) (Figure 2 ) (Schneider et al., 2012 (link)), and the analysis of morphological features was performed with CellSegm, a MATLAB (MathWorks, Natick, MA) segmentation toolbox (Hodneland et al., 2013 (link)), and represented using the UCSF Chimera package (Pettersen et al., 2004 (link)).
A22284
Manufactured by Thermo Fisher Scientific
The A22284 is a laboratory equipment product from Thermo Fisher Scientific. It serves as a core function in laboratory settings, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
V9131, by Merck Group (1 mentions)
Ht501128, by Merck Group (1 mentions)
Nucblue, by Thermo Fisher Scientific (1 mentions)
A21202, by Thermo Fisher Scientific (1 mentions)
Matlab, by MathWorks (1 mentions)
Vibratome, by Leica camera (1 mentions)
A11010, by Thermo Fisher Scientific (1 mentions)
D3571, by Thermo Fisher Scientific (1 mentions)
A21428, by Thermo Fisher Scientific (1 mentions)
Vectashield h 1000, by Vector Laboratories (1 mentions)
A34055, by Thermo Fisher Scientific (1 mentions)
A21121, by Thermo Fisher Scientific (1 mentions)
A 21311, by Thermo Fisher Scientific (1 mentions)
G1546, by Merck Group (1 mentions)
Sc 576, by Santa Cruz Biotechnology (1 mentions)
Lsm 880, by Zeiss (1 mentions)
Triton x 100, by Merck Group (1 mentions)
4 protocols using a22284
1
Quantitative Analysis of Cell Morphology
2
Peptide Gel Immunostaining Protocol
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Peptide gels were washed twice in PBS before fixation with 4% paraformaldehyde in PBS. The fixed gels were washed and stored in PBS, before embedding in 2% agar to allow thick sectioning using a vibratome (Leica, section thickness 500 µm).
Each gel section was covered with blocking buffer (0.5% bovine serum albumin in PBS + 0.1% triton) for 30 min prior to staining. For laminin staining, the gels were incubated overnight in a solution of primary antibody (abCam ab11575) at 1:100 dilution in blocking buffer. The gel sections were then washed in PBS and incubated overnight with secondary antibody (Invitrogen a11010) and phalloidin (F432 Thermofisher), both at 1:400 dilution. For cytokeratin 18 staining, gels were incubated overnight with 200 μl of pre-conjugated antibody at 1:100 (Thermofisher 53–9815-82) along with Alexa Fluor 633 conjugated phalloidin at 1:50 (A-22284 Invitrogen).
The gels were washed twice with PBS then incubated for 1 h in 1:1000 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen D3571) before imaging.
Each gel section was covered with blocking buffer (0.5% bovine serum albumin in PBS + 0.1% triton) for 30 min prior to staining. For laminin staining, the gels were incubated overnight in a solution of primary antibody (abCam ab11575) at 1:100 dilution in blocking buffer. The gel sections were then washed in PBS and incubated overnight with secondary antibody (Invitrogen a11010) and phalloidin (F432 Thermofisher), both at 1:400 dilution. For cytokeratin 18 staining, gels were incubated overnight with 200 μl of pre-conjugated antibody at 1:100 (Thermofisher 53–9815-82) along with Alexa Fluor 633 conjugated phalloidin at 1:50 (A-22284 Invitrogen).
The gels were washed twice with PBS then incubated for 1 h in 1:1000 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen D3571) before imaging.
3
Immunodetection of Myogenic Markers in Zebrafish Larvae
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Larvae were fixed with 2% paraformaldehyde in PBS for 25 min to overnight depending on the stage. Immunodetection for slow myosin heavy chain (MyHC) (1:5, F59; Devoto et al., 1996 (link)), general MyHC (1:10, A4.1025; Blagden et al., 1997 (link)), Pax7 (1:5; DSHB; Kawakami et al., 1997 (link)), Myogenin (1:50, sc-576, Santa Cruz) and GFP (1:500, TP-401, Torrey Pines or 1:500, G1546, Sigma) was performed in PBS with 0.5-1% Triton X-100 (PBT) for between overnight and 5 days at 4°C on a rotary shaker, depending on larval age and antigen (Hinits and Hughes, 2007 (link)) followed by Alexa Fluor 488 or 555 secondary antibodies (1:1000; A21121 and A21428, respectively; Invitrogen). at least overnight (±Hoechst 33342) at 4°C. After EdU detection in Fig. S4 , samples were blocked in 5% BSA, 3% normal goat serum, PBT for 20 min, incubated using Alexa Fluor 488-conjugated anti-GFP (1:500, A-21311, Molecular Probes) and Hoechst in block buffer (shaking at 4°C for 3-6 h), followed by 6×15 min washes in PBT. Phalloidin-Alexa Fluor 555 or phalloidin-Alexa Fluor 633 (1:1000, A34055 or A22284, Thermo Fisher) were used to-stain F-actin. Larvae were mounted on glass slides under bridged coverslips in Citifluor AF1 or Vectashield (H-1000, Vector Laboratories). In situ mRNA hybridisation was as described (Groves et al., 2005 (link)).
4
Immunofluorescence Imaging of Tight Junction Proteins
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Animals were euthanized with an overdose of sodium pentobarbital and decapitated. Temporal bones were quickly removed, and the cochleae were treated with intrascalar perfusion with 4% paraformaldehyde in PBS (ChemCruz, Santa Cruz Biotechnology, Inc., Dallas, TX). Cochleae were fixed in 4% paraformaldehyde in PBS for 1 h at room temperature (RT) and then rinsed with PBS. The RWM was carefully dissected under a dissecting microscope and immersed in the same fixative at 4°C overnight. Postfixed RWMs were permeabilized using 0.1% Triton X-100 (Sigma) in PBS for 40 min at RT and then rinsed thoroughly in PBS. Tissues were incubated with primary mouse monoclonal anti-ZO1 antibodies (1:100; Thermo Fisher Scientific, Waltham, MA) and rabbit monoclonal antioccludin antibodies (1:100; Abcam Inc., Cambridge, United Kingdom). After three 10 min washes with PBS, tissues were incubated with Alexa Fluor 488–labeled donkey antimouse antibodies and 555-labeled goat antirabbit antibodies (1:500; Thermo Fisher Scientific) for 2 h at RT. Following incubation with Alexa Fluor 633–labeled phalloidin (1:100; A22284, Thermo Fisher Scientific) for 30 min, samples were mounted in 4,6-diamidino-2-phenylindole (DAPI) Fluoromount-G mounting medium and covered with a coverslip. The fluorescence images were obtained with a confocal laser scanning microscope (Zeiss LSM 880, Carl Zeiss).
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