Protein Methods Articles:
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Enhanced immunostaining means more signal and better sensitivity
Achieve better protein detection using a fraction of your valuable primary antibody.
Boost the performance of your antibody with Thermo Scientific Pierce Immunostain Enhancer. The enhancer enables significant antibody dilution (5- to 20-fold beyond vendor recommendations) without adding additional time to the immunostaining procedure. The ready-to-use enhancer does not add steps to your protocol because it is used to dilute the primary and secondary antibodies.
The Pierce Immunostain Enhancer is compatible with chromogenic (Figure 1) and fluorescent (Figures 2-3) detection and routinely increases both signal intensity and detection sensitivity. Signal enhancement is antibody-dependent and typically ranges from 3- to 12-fold. Because of the increased signal intensity, less antibody is required to achieve optimal detection.
Figure 1. Improve specificity and signal intensity and reduce background in chromogenic immunohistochemistry. Cytokeratin is detected in poorly differentiated colon adenocarcinoma tissue section using rabbit anticytokeratin 18 antibody at 0.01µg/mL and HRP-conjugated goat anti-rabbit IgG (Part No. 31460). The antibodies were diluted with either bovine serum albumin (BSA) blocking buffer or Pierce Immunostain Enhancer. Detection was performed using Thermo Scientific Metal Enhanced DAB Substrate Kit (Part No. 34065).
Figure 2. Increased sensitivity and signal intensity for fluorescent detection of specific targets. Ezrin is detected in A549 cells using anti-ezrin mouse monoclonal antibody and goat anti-mouse IgG conjugated to Thermo Scientific DyLight 488 Dye (Part No. 35502). Ezrin antibody specifically stains microvillar peripheral membrane protein in the cytoplasm. Panel A: Detection of ezrin in BSA blocking buffer (dilution 1:200). Panel B: Detection using 25-times less ezrin antibody (1:1250 dilution). Images were acquired at the same gain and exposure time using appropriate optical filter sets with a 20X (0.45 NA) objective.
Figure 3. Obtain better images using less primary antibody (200-fold dilution of lamin B1 primary antibody beyond the vendor’s recommendation). Panel A: Lamin B1 is detected in A549 cells using a 1:125 dilution of the primary antibody in BSA; Panel B: The same dilution results in oversaturation of the signal when the Pierce Immunostain Enhancer is used. Panel C: The antigen is undetectable when the primary antibody is diluted 1:25,000 in standard buffer; Panel D: Pierce Immunostain Enhancer results in clear detection of lamin B1 at the same dilution as Panel C. Images were acquired at the same gain and exposure time using appropriate optical filter sets with a 20X (0.45 NA) objective.
Formalin-fixed paraffin-embedded tissue sections were deparaffinized then subjected to heat-induced epitope retrieval using citrate buffer and endogenous enzyme quenching and blocking. A PAP pen was used to delineate the tissue sections. The primary and the secondary antibodies were diluted with either 2% Thermo Scientific Blocker BSA (Part No. 37525) or the Pierce Immunostain Enhancer. The primary antibody was incubated for one hour at room temperature or overnight at 4°C. The tissues were incubated with HRP-conjugated secondary antibody for 45 minutes at room temperature and detected using Metal Enhanced DAB Substrate Kit (Part No. 34065).
A549 cells were seeded at 5000 cells per well in a 96-well plate and incubated for 18-20 hours at 37°C, 5% CO2 in a humidified incubator. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Thermo Scientific Surfact-Amps X-100 and then blocked with 2% BSA, 0.1% Triton* X-100 at room temperature for 30 minutes. The primary and the secondary antibodies were diluted with either BSA blocking buffer or the Pierce Immunostain Enhancer. The cells were incubated with primary antibody for one hour at room temperature or overnight at 4°C followed by Thermo Scientific DyLight Dye-conjugated secondary antibodies for one hour at room temperature.
The images were acquired with a 20X (0.45 NA) objective at the same gain and exposure time using appropriate optical filter sets on the Axio Observer*. Z1 Microscope (Carl Zeiss Inc.) using either an ORCA-ER-1394 CCD digital camera (Hamamatsu) or an Axio MRC3 color digital camera.