Mayer s haematoxylin

10% formalin fixed adult female worms embedded in paraffin (Histology Laboratory, Washington State University) were sectioned, attached to glass slides and deparaffinized and steam treated. To assess specificity of ConA binding, sections were treated with sodium periodate (5 mM in 50 mM sodium acetate buffer, pH 4.5), followed by sodium borohydride (50 mM in PBS, pH 7.4) to disrupt carbohydrates containing vicinyl hydroxyl groups. Slides were ten treated with 0.3% hydrogen peroxide in methanol for 30 min at 25°C to eliminate endogenous peroxidases and then incubated with ConA-horse radish peroxidase (HRPO). Binding was localized by development with Metal Enhanced DAB Substrate (Thermo Scientific, Rockford, IL.). Sections were then counterstained with Mayer's haematoxylin (Thermo Scientific, Fremont, CA).

Testes were fixed in neutral buffered formalin (NBF) for 24 h, transferred to 70% ethanol, machine processed and paraffin embedded. Formalin-fixed paraffin-embedded (FFPE) sections of 3 µm thickness were used for all histological stains and immunohistochemistry (IHC).
For Periodic Acid Schiff (PAS) stainings slides were dewaxed, washed in water and placed in 0.5% Periodic Acid (Sigma P0430) for 5 min. After three washes in ultra-pure water, slides were placed in Schiff reagent (Thermo Fisher Scientific, J/7300/PB08) for 15–30 min in a closed container and washed again three times in ultra-pure water. Counterstain was performed using Mayers Haematoxylin (Thermo Fisher Scientific, LAMB/170-D) for 40 s followed by rinsing in tap water, dehydration and mounting.
IHC was performed on FFPE sections using the Bond™ Polymer Refine Kit (DS9800, Leica Microsystems) on the automated Bond Platform. Anti-phospho-Histone H3 (Ser10) (pH3) antibody (Upstate, 06-570, 1:200 dilution) was used with DAB Enhancer (Leica Microsystems, AR9432) and heat-induced epitope retrieval was performed for 10 min at 100 °C on the Bond platform with sodium citrate. All slides were scanned using Aperio XT (Leica Biosystems) and PH3 intensities were quantified using the Aperio eSlide Manager (Leica Biosystems).

For immunohistochemistry stainings, FDA standard tissue array and tumor tissue array (Tissue Array) were deparaffinized and rehydrated in a descending alcohol series before antigen unmasking with TrisEDTA (pH 9.0). Endogenous peroxidase was blocked using 3% H₂O₂ followed by non-specific binding block with BlockAid (Invitrogen). The tissues were then stained with Nemod-TF2 (Glycotope) and Envision Flex (Agilent), and the color developed using DAB+ staining solution (Agilent). All slides were counterstained using Mayer’s Haematoxylin (Thermo Fisher Scientific) and mounted in Entellan (Merck). Slides were photographed using VS200 Research Slide Scanner (Olympus Lifescience) and evaluated by two independent analysts to determine the immunoreactive score (IRS, score range 0-12) (20 ) for presence of membranous CD176 on tumor and H-Score (score range score range 0-300) (21 (link)) for presence on normal tissues.