Horseradish peroxidase hrp

IHC expression of ER, PR, HER2, EGFR, and CK5/6 were assessed on triplicate tissue microarrays (TMA) and reviewed by two pathologists. The tissue was formalin-fixed, processed and paraffin-embedded using routine protocols. Three-micron sections were cut and stained with hematoxylin and eosin (Dako). Standard IHC protocols were used. Antigen retrieval was carried out using citric acid buffer pH 6 (Dako). For visualization of nuclei, hematoxylin counterstain was used. Secondary antibodies conjugated to horseradish peroxidase (HRP) (Dako) were used for visualization with 3,3-diaminobenzidine (DAB) (Dako), according to manufacturer’s protocol.

HUVECs were lysed with Radioimmunoprecipitaion assay (RIPA) buffer (Thermo Fisher) supplemented with Halt™ Protease and Phosphatase Inhibitor Cocktail (1:100; Thermo Fisher). Cell lysis was enabled by an incubation at 4°C at a turning wheel for 1 h. Cell debris was removed by centrifugation (16000 xg for 10 min at 4°C). Protein concentration was determined by Pierce^™ BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of denaturated protein in Laemmli buffer were loaded on 12% Sodium dodecyl sulfate gels (BioRad) and blotted on nitrocellulose membranes (Invitrogen). Membranes were blocked with 3% milk (Roth) in TBS-T and incubated with primary antibody overnight at 4°C under rotation. Secondary antibodies tagged with Horse Radish Peroxidase (HRP; Dako) were incubated for 1 h at room temperature under rotation. Bands were visualized using enhanced chemiluminescence (ECL, Thermo Fisher) on the ChemiDoc device (BioRad). Band intensity was quantified using ImageLab Software (version 5.2.1; BioRad). Antibodies and dilutions can be found in the S1 Table and uncropped images of Western blots are available in S1 Raw image.

(i) Western blot analysis of HIV-1 Env-mediated α-tubulin acetylation was studied in CEM.NKR-CCR5 cells (1 × 10⁶ cells) incubated with 500 ng of p24 of luciferase reporter pseudoviruses for 1 h at 37°C (33 (link)). Membranes were probed with the anti-α-tubulin B-5-1-2 monoclonal antibody (MAb) and the anti-acetylated α-tubulin 6-11B-1 MAb (both from Sigma-Aldrich, St. Louis, MO) and secondary antibodies conjugated with horseradish peroxidase (HRP) (Dako, Glostrup, Denmark). The increase in α-tubulin acetylation was quantified and expressed as the ratio of the intensities of the acetylated α-tubulin to the total α-tubulin bands, as described previously (33 (link), 48 (link)). (ii) Immunofluorescence of HIV-1 Env-mediated acetylated α-tubulin and the F-actin capping assay was analyzed in CEM.NKR-CCR5 cells (1 × 10⁶ cells/point) incubated with HIV-1 luciferase reporter pseudoviruses (29 (link), 30 (link), 33 (link)), as described for Western blot analysis. Coverslips were mounted in Mowiol antifade (Dako) and imaged in xy midsections in a FluoView FV1000 confocal microscope through a 1.35 NA objective (60×) (Olympus, Center Valley, PA) for high-resolution imaging of fixed treated cells. The final images and molecule codistributions were analyzed and quantified with MetaMorph software (Universal Imaging, Downington, PA).

Paraffin-embedded sections were deparaffinized in xylene and rehydrated through a series of decreasing concentrations of ethanol. Staining was carried out using indirect immunoperoxidase diaminobenzidine (DAB). Endogenous peroxidase was blocked by 0.3% H₂O₂ for 5 min. Sections were incubated for 1 h at room temperature with polyclonal anti-PCNA (1:200), NF-κB p50 (1:500), HMGB1 (1:200) and VEGF Ab (1:200) in Ab diluent with background reducing components (DakoCytomation, Carpinteria, CA, USA). As a negative control, the IgG isotype control was employed at the same time and concentration as the test antibodies. After rinsing with PBS, sections were finally developed with a DAKO LSAB+ System, horseradish peroxidase (HRP) (DakoCytomation; KO679), and immunostaining was visualized with substrate solution (DAB). Counterstaining was performed with Mayer’s hematoxylin. Immunostaining of PCNA and NF-κB p50 were localized in the nucleus of epithelial cells. VEGF staining was noticed in the cytoplasm. The number of PCNA and NF-κB p50 immunopositive cells from six slides in experimental and control samples were selected randomly and evaluated by two blinded observers and scored.

After protein, denatured proteins were separated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis on acrylamide gel, and transferred to Hybond membranes. The membranes were blocked overnight in 5% skim milk in TBST. For immunoblotting, the membranes were incubated with the primary antibody (Table 3), rinsed with TBST, and incubated with IgG antibodies conjugated to horseradish peroxidase (HRP; Dako). Bands were visualized using X-ray film by ECL-Plus detection reagents. Densitometric quantification of acetyl-histone 3 and 4 protein expression in gastric samples was performed using Image J software, with GAPDH as a control.