Dab quanto

Tissues were fixed by 4% paraformaldehyde overnight, embedded in paraffin and cut in 5 μm for experiments. Tissue sections were deparaffinized, hydrated, and heated in a steamer for antigen retrieval. The tissue samples were then incubated overnight with rabbit anti-human Ku80 monoclonal antibody (Thermo Fisher Scientific, Fremont, CA, USA) at 1:500 dilution, followed by incubation with goat anti-rabbit secondary antibody (Thermo Fisher Scientific, Fremont, CA, USA). The sections were washed extensively and incubated with DAB Quanto (Thermo Fisher Scientific, Fremont, CA, USA) for 5 min. The sections were stained, fixed, and visualized. Each section was examined by two pathological experts independently who were blinded to the clinical data. The immunohistochemical score (IHS) was obtained by combination of proportion score (average percentage of the five fields) with intensity score of the stained tumor cells. Staining intensity was divided into 3 level: 0 (negative), 1 (weak staining), 2 (moderate staining) and 3 (strong staining). The proportion of the tumor cells were as follow: 0 = 0 ~ 5%, 1 = 5 ~ 10%, 2 = 11 ~ 49% and 3 ≥ 50%. The final IHS ranged from 4 to 9 was considered positive staining [10 (link)].

The tissue slides were deparaffinized in dimethylbenzene, rehydrated through graded alcohols, and incubated in acidic (R&D) and Hot Rinse (Biocare Medical) for antigen retrieval in the Retriever 2100 (Proteogenix). After washing in running water, the slides were immersed in 3% hydrogen peroxide in methanol for 10 minutes to block endogenous peroxidase. After incubation in 5% goat serum, the sections were incubated overnight at 4°C in primary antibody diluted in 5% goat serum. APPL1 antibody (Cell Signaling) was used at a 1:100 dilution. After washing with PBS, the slides were incubated with secondary antibody (Envision^™ System, Dako) for 30 minutes at room temperature. The sections were washed and developed with DAB Quanto (Thermo Scientific) under microscopic control. The reaction was stopped with tap water, and the sections were stained with hematoxylin, dehydrated, and mounted. Images were acquired with Pannoramic 250 Flash. An ethical permit to use tumour tissues for generation of tissue slides was provided by the Umeå Ethical Review Board in full agreement with the Swedish Ethical Review Act (540/03, Dnr 03–482).

BC paraffin-embedded tissues were sectioned at 4 μm, after which they were stained with rabbit anti-human GPR68 antibody (Invitrogen, USA, Cat. no: 720277), at 1:200 dilution. The secondary conjugation and detection were done using UltraVision Quanto Detection System HRP and DAB Quanto (Thermofisher Scientific, USA). The images were captured with Olympus DP74 microscope digital camera attached to a BX43 microscope (Olympus Life Sciences, Tokyo, Japan). Immunoreactive score (IRS) was used to evaluate the expression status of GPR68 in the different samples according to the recommendations by Remmele and Stegner (24 ). IRS is usually generated by the multiplication of the staining intensity and the percentage of immuno-stained cells with a range from 0-12. Microscopic evaluation of the immunohistochemical stainings was performed by two independent investigators. Also, semiquantitative analysis of DAB staining of GPR68 was done using the immunohistochemistry (IHC) Toolbox plugin in Image J software (https://imagej.nih.gov/ij/index.html). Optical density (OD) was calculated as log (max intensity/mean intensity).

Standard protocols were used to prepare tissue microarray sections for immunohistochemistry. Then sections were blocked by UltraVision Hydrogen Peroxide Block (Thermo Scientific, CA, USA) and UltraVision Protein Block (Thermo Scientific), followed by primary antibodies (VEGF, 1:100) incubation. UltraVision Quanto Detection System horseradish peroxidase (HRP) Polymer (Thermo Scientific) and DAB Quanto (Thermo Scientific) were applied for staining, and hematoxylin was used for counterstaining. The score of erythema, scale and infiltration thickening of the skin lesions in mice was scored as 0 to 4: 0, negative; 1, weak; 2, moderate; 3, strong; and 4 very strong. Add the three scores to get the total points, taking the average of the scores of the mice in each group, and skin lesions in each group were observed.

T. spiralis ML harvested at 35 days post-infection were fixed by ice-cold methyl alcohol and incubated in Triton-100 overnight. Worms were then incubated with anti-rTsTPX2 antibodies and Alexa Fluor^® 488 labeled secondary antibodies. Stained T. spiralis ML were imaged with a confocal laser scanning microscope (Leica TCS SP8).
Small intestines harvested at 3 days post-infection and diaphragms harvested at 35 days post-infection from T. spiralis infected BALB/c mice were fixed in 4% paraformaldehyde. Thin sections of the embedded tissues were first stained with anti-rTsTPX2 antibodies and then incubated with Alexa Fluor^® 488 labeled secondary antibodies or DAB Quanto (Thermo Fisher Scientific). Confocal images of stained tissues were obtained with a confocal laser scanning microscope (Leica TCS SP8).