After the transfer, blocking of unspecific binding sites was achieved by incubation in TBST (50 mM Tris/HCl, 150 mM NaCl, 0.5% Tween 20, pH 7.2) containing 5% skimmed milk.
With improved fluorochrome labeling of dsDNA, removal of DNA aggregates, and enhanced blocking of unspecific binding, we were able to specifically detect dsDNA-reactive plasma cells by immunofluorescence microscopy.
After washing the plates and blocking of unspecific binding sites, c-jun was detected using an ELISA detection reaction.
Slides were washed 5 times with PBS prior to blocking of unspecific antibody binding with DAKO protein block serum-free (Dako, Hamburg, Germany) for 20 min at 37°C.
After blocking of unspecific binding sites with 1% BSA in PBS at room temperature for 1 h, staining with anti-SAMHD1 antibody (ProteinTech Group, Chicago, IL, USA) was performed.
In brief, paraffin was removed followed by blocking of endogenous peroxidase activity, heat-induced epitope demasking and blocking to prevent unspecific binding.
For double-labeling with phalloidin and vinculin, the first labeling was performed with a 20-min incubation with Alexa 488 phalloidin (1 40 dilution; Invitrogen), followed by block of unspecific binding sites and then by overnight incubation with a monoclonal antibody to vinculin (1 100 dilution; Sigma-Aldrich).
Blocking of unspecific sites was achieved by incubation with 10% donkey or goat serum (Sigma-Aldrich, St . Louis MO, USA) for 1 h at room temperature.
PaTu-S and PaTu-T cells grown on glass coverslips for 24 48 h were washed 3 times with PBS, fixed for 15 min in 4% paraformaldehyde and permeabilised in 0.1% Triton- X-100 for 5 min. Blocking of unspecific binding was achieved by a 1 h incubation in 10% Aurion BSA-c (Aurion, Waageningen, The Netherlands).
Blocking of unspecific binding sites was achieved by incubation of the membrane in 5% low fat milk powder in PBS/0.2% Tween-20 (blocking buffer) for 1 hour at room temperature.
