Cytospins were prepared with CD34+ cells, air-dried and fixed with buffered 10% formalin. The cytospins were briefly washed with Tris-buffered saline with 0.1% Tween (which was also used throughout the procedure in washing steps) to remove formalin residue and then subjected to heat-induced epitope retrieval process using Diva buffer (Biocare Medical, CA) and a Decloaking Chamber (pressure cooker) (Biocare Medical, CA). The cytospins were incubated with an anti-IL11R mouse monoclonal antibody at a dilution of 1:10 (4D12; Santa Cruz Biotechnology, CA) at room temperature in a dark chamber for 1h. Detection of the primary antibody was achieved with the Mach4 kit (Biocare Medical, CA) used according to the manufacturer’s instruction. Chromogen development was performed with DAB+ (DakoCytomation). Slides were counterstained with hematoxylin, air-dried and cover-slipped.
Diva buffer
Manufactured by Biocare Medical
Diva buffer is a general-purpose buffer solution used in various laboratory applications. It is designed to maintain a stable pH environment for biological samples and reactions.
Automatically generated - may contain errors
Lab products found in correlation
Decloaking chamber, by Biocare Medical (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
A21240, by Thermo Fisher Scientific (1 mentions)
Hoechst dye, by Thermo Fisher Scientific (1 mentions)
Multispectral imaging system, by Nikon (1 mentions)
Mca1477, by Bio-Rad (1 mentions)
2 protocols using diva buffer
1
Immunohistochemical Detection of IL11R
2
Immunofluorescent Staining of Tissue Sections
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Slides were deparaffinized through a graded alcohol series, and antigen retrieval was performed using a decloaking chamber (Biocare medical, #DC2002) at 125°C for 3 minutes using Diva buffer (Biocare medical, #DV2004G1). Slides were blocked for 1 hour using 0.2% bovine serum albumin + 2% normal goat serum, incubated with primary antibodies (CD3 clone CD3-12, bio-rad #MCA1477, Granzyme B, rabbit polyclonal, LSBio # 34084), diluted 1:500 each for 1 hour in blocking buffer for 1 hour, RT. Slides were washed in PBS, and incubated in secondary antibody (Fisher #A-21240) diluted 1:500 in blocking buffer for 1 hour, RT. Slides were washed in ddH2O, and incubated with Hoechst dye (Fisher, #H3569) diluted to 2 μg/ml in ddH2O for 10 minutes. Slides were imaged on a Nuance Multispectral Imaging system on a Nikon Eclipse Ci microscope.
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