Cells were fixed in 4% paraformaldehyde (PFA) for 15 min at room temperature (RT) and washed twice with 1× PBS before being permeabilized with 0.2% Triton X-100 in 1× PBS for 10 min. Cells were rinsed twice in 1% bovine serum albumin (BSA) in 1× PBS and blocked for 30 min at RT with DAKO blocking buffer (Agilent). Cells were incubated with primary antibodies in 1% BSA at 4 °C for O/N, and then washed twice and incubated with the appropriate secondary antibody for 1 h at RT (Table 3 ). Finally, cell nuclei were stained with DAPI (1:1000) at RT for 5 min, washed twice and visualized and captured using an Olympus IX73 inverted microscope connected to a XM10 monochrome camera (Olympus, Tokyo, Japan).
Dako blocking buffer
Manufactured by Agilent Technologies
Sourced in United States
DAKO blocking buffer is a laboratory reagent used to prevent non-specific binding in immunohistochemical and related techniques. It is designed to reduce background staining and improve the specificity of antibody-based detection methods.
Automatically generated - may contain errors
Lab products found in correlation
Xm10 monochrome camera, by Olympus (2 mentions)
Ix73 inverted microscope, by Olympus (2 mentions)
Rabbit anti lc3a b, by Cell Signaling Technology (1 mentions)
Dako antibody diluent, by Agilent Technologies (1 mentions)
Guinea pig anti insulin, by Bio-Rad (1 mentions)
Rabbit anti p62, by Abcam (1 mentions)
Bloxall, by Vector Laboratories (1 mentions)
Dab solution, by Vector Laboratories (1 mentions)
Anti androgen receptor, by Santa Cruz Biotechnology (1 mentions)
Anti c myc, by Abcam (1 mentions)
4 protocols using dako blocking buffer
1
Immunocytochemical Staining Protocol
2
Immunocytochemical Staining Protocol
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Cells were fixed in 4% paraformaldehyde (PFA) for 15 min at room temperature (RT) and washed twice with 1× PBS before being permeabilized with 0.2% Triton X-100 in 1× PBS for 10 min. Cells were rinsed twice in 1% bovine serum albumin (BSA) in 1× PBS and blocked for 30 min at RT with DAKO blocking buffer (Agilent). Cells were incubated with primary antibodies in 1% BSA at 4 °C for O/N, and then washed twice and incubated with the appropriate secondary antibody for 1 h at RT (Table 3 ). Finally, cell nuclei were stained with DAPI (1:1000) at RT for 5 min, washed twice and visualized and captured using an Olympus IX73 inverted microscope connected to a XM10 monochrome camera (Olympus, Tokyo, Japan).
3
Immunofluorescence Analysis of Cellular Processes
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Tissue sections were dewaxed in xylene and hydrated in serial dilutions of ethanol (2×; 100%, 90% and 70%) followed by water. Heat- and citrate-based antigen retrieval was performed using Vector Laboratories (CA, USA) antigen unmasking solution (H-3300) for 20 min and then the slides were allowed to cool to room temperature for 1 h. Slides were blocked with Dako blocking buffer (Agilent Technologies, CA, USA, #X0909) prior to incubation with antibodies in Dako antibody diluent (Agilent Technologies #S3022). Antibodies and the corresponding dilutions used are listed as follows: Developmental Studies Hybridoma Bank (DSHB, Iowa, USA) mouse anti-Proinsulin (GS-9A8; 1:50), rat anti-lysosomal-associated membrane protein 1 (LAMP1) (DSHB 1D4B; 1:50), rabbit anti-LC3A/B (Cell Signaling Technology, MA, USA, #12741; 1:100), rabbit anti-p62 (Abcam, Cambridge, UK, #ab91526; 1:200) and guinea pig anti-insulin (BioRad, CA, USA, #5330-0104G; 1:500). Highly cross-adsorbed fluorescently conjugated secondary antibodies were used. Images were collected on either a Zeiss (Carl Zeiss, Oberkochen, Germany) LSM 700 confocal microscope using a 63X/1.4 numerical aperture oil objective, or a Zeiss LSM 800 confocal microscope equipped with Airyscan using a 63X/1.4 numerical aperture oil objective.
4
Immunohistochemical Analysis of c-Myc and AR
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Immunohistochemistry was performed as previously described [22 (link)]. Briefly, heat-induced antigen retrieval was carried out in a steam chamber followed by washing in Tris-buffered saline. Endogenous peroxidases were inactivated with Bloxall (Vector labs), and sections were blocked with Dako blocking buffer (Agilent Technologies), incubated with primary antibodies (anti-c-Myc, anti-AR), washed, and then incubated with horse radish peroxidase-conjugated secondary antibodies. Chromagen was developed with DAB solution (Vector Labs) and counterstained with Meyer’s hematoxylin. Primary antibodies used were anti-cMyc (Abcam, clone 769, 1:1000), anti-Androgen Receptor (Santa Cruz, clone N-20, 1:500), and anti-CK8 (Covance, clone 1E8, 1:1000).
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