Vector immpact dab

Immunostaining was performed following deparaffinization and rehydration of the slides, antigen retrieval was performed in a pressure cooker using citrate buffer (pH 6.0) for 4 min. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 15 min at room temperature and nonspecific binding was blocked using 10% normal horse serum for 30 min at room temperature. Samples were incubated at room temperature with p-Y416Src rabbit monoclonal antibody (Y416), cleaved caspase 3, p-Y705Stat3 and Ki67. Slides were developed with Vector Immpress rabbit IgG (#MP7401) and Vector Immpress mouse IgG (#MP7400) for 30 min at room temperature. Chromogenic detection was performed using Vector Immpact DAB (#SK4105) for 3 min. Sides were counterstained with hematoxylin. A 3DHistech MIDI Scanner (Perkin Elmer) was used to capture whole slide digital images with a 20x objective. Images were converted to into MRXS files and computer graphic analysis was completed using inForm 1.4.0 Advanced Image Analysis Software (Perkin Elmer).

7μm frozen sections were dried overnight then fixed in a 3:1 mix of anhydrous acetone/ethanol for 5 minutes. Endogenous peroxidase was blocked using 0.5% (v/v) hydrogen peroxide in methanol for 10 minutes and non-specific binding of the primary antibody minimized by incubating sections in Biocare Medical Background Sniper plus 2.0% (w/v) BSA for 10 minutes. The primary antibody, hamster anti-mouse LV9 diluted 1:1000 in Biocare Medical Da Vinci Green antibody diluent, was applied for 60 minutes then detected by applying Jackson Immunoresearch goat anti-hamster secondary, diluted 1:300 in TBS, for 30 minutes followed by a 30 minute application of Vector ImmPRESS Goat HRP polymer. LV9 signal was visualized using Vector ImmPACT DAB. Sections were counterstained with Mayer’s Haematoxylin in a Leica Autostainer XL and mounted using a Leica CV5030 coverslipper.

The tumor specimens were processed with the use of alcohols and xylene and then infiltrated in paraffin wax using the Excelsior ES Tissue Processor. Paraffin sections were dewaxed in xylene, rinsed in grade alcohol, and rehydrated in water and then were placed in citric buffer (PH 6.0) and treated in a microwave oven with high power for 3 min and 10% goat serum for 30 min. Subsequently, antibodies with proper dilution were applied on the sections as follows: CD31 (Pierce Product# PA5-32321; 1∶50 dilution, 1 h at RT) and Ki-67 (Cell Signaling Product# #9027; 1∶150 dilution, 1 h at RT). Following that, secondary antibodies (Ventana Multimer Anti Rb-HRP Product#760-4311 24 min at RT) were applied. Signals were developed with Vector ImmPACT DAB Product#SK-4105 for 8 mins at RT. The sections were finally counter stained by hematoxylin solution for 1 min at RT.

Paraffin-embedded tissue sections (4 μm) were dewaxed in xylenes 3 times, passed through a series of graded alcohols, and washed in deionized water. For antigen retrieval, 1X CitraPlus AR Buffer (Biogenex) was pre-heated for 20 min in an Oster steamer before adding the slides for an additional 20 min. After cooling for 10 min, slides were washed 3 times in deionized water. Sequential blocking steps were performed—10 min in Bloxall (Vector Laboratories), 30 min in F_c Receptor Block (Biogenex) and 30 min in 1% normal horse serum (Vector Laboratories). Sections were incubated overnight at 4 °C in 1:200 rabbit IgG anti-CDKN2A/p16INK4a antibody (Abcam, ab108349). After washing 3 times in TBST, sections were incubated for 30 min in Vector ImmPRESS HRP Horse Anti-Rabbit IgG Polymer reagent. After washes, slides were developed for 3 min with Vector ImmPACT DAB. Sections were then counterstained with hematoxylin, dehydrated, cleared and mounted using VectaMount (Vector Laboratories). Images were captured using a Leica DM 2000 upright microscope.

Brain samples from sacrificed rats were collected and fixed in 10% neutral buffered formalin. Tissues were processed with increasing ethanol series (70, 80, 95, and 100%), and they were treated with xylene and embedded in paraffin blocks. A Thermo HM355S microtome was used to section tissues at 5 μm thickness. Hematoxylin and eosin (H&E) staining was performed according to the standard protocol. For immunohistochemistry experiments, sections were labeled with anti-IBA-1 antibody (1:8000; Abcam, ab178847) and horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5000; Vector Labs, MP-745). Vector ImmPACT DAB (Vector Labs, SK-4105) was applied for 5 min, and hematoxylin counterstaining was performed. Digital images were acquired at 100X total magnification using an Olympus BX43 microscope equipped with LCmicro v2.2 software.