Bsa blocking buffer

96-Well plates (Nunc, Maxisorp) were coated with, native Insulin (Sigma-Aldrich, Cat. 91077C), Streptavidin (ThermoScientific, Cat. 21125), calf thymus DNA (ThermoScientific, Cat.15633019), or NP-BSA (Biosearch Technologies, N-5050H-100) with 10 µg/mL, or anti-IgM, anti-IgG-antibodies (SouthernBiotech). Biotinylated peptides (2,5 µg/mL) were loaded onto SAV-coated plates in 1% BSA blocking buffer (ThermoFisher). Sera were initially diluted 1:50. Serial dilutions of 1:3 IgM or IgG antibodies (SouthernBiotech) were used as standard. The relative concentrations, stated as arbitrary unit (AU), were determined via detection by Alkaline Phosphatase (AP)-labeled anti-IgM/anti-IgG (SouthernBiotech), respectively. The p-nitro-phenylphosphate (pNPP; Genaxxon) in Diethanolamine buffer was added for starting of the reaction. Data were acquired at 405 nm using a Multiskan FC ELISA plate reader (Thermo Scientific). Samples were measured at least in duplicates.
For analysis of affinity-maturation (22 (link)), results from plates coated with InsA(1) or InsA(4) were calculated by dividing InsA(1) by InsA(4). Here, we used Streptavidin with tetra- (ThermoScientific) or monovalent binding capacity. Subsequently, results were stated as relative units [RU].

SKBR3 cells (ATCC,
HTB-30) and MCF7 cells (ATCC, HTB-22) were seeded at the density of
7,000 cells per well in a 96-well glass-bottom plate (Cellvis, P96-0-N)
and incubated for 3 days before running the assay to reach the approximate
confluency of 70%. After 3 days, the medium was removed, and the cells
were fixed using 4% Paraformaldehyde (Alfa Aesar, J62478), washed
3 times (5 min each), and blocked using 2% BSA blocking buffer (Thermo
Scientific 37525) for 1 h. The antibody dye conjugates (Fluorescein-HER2
Ab as the positive control and different TPE-HER2 Ab conjugates) were
diluted to the concentration of 10 μg/mL in 0.1% BSA solution,
added to the fixed SKBR3 and MCF7 cells, and left at 4 °C overnight.
The samples were washed with 1× PBS 2 times and imaged on a Zeiss
Axio Observer connected to an X-cite Series 120Q light source using
the excitation and emission filters of interest using a 20× objective.
The TPE filter cube has an excitation of G365, BS of 395, and emission
BP of 535/30. The Fluorescein filter cube has an excitation BP of
500/25, BS of 515, and emission BP of 535/30.