Envision flex target retrieval solution

The study was based on tumors obtained from 60 patients with serous ovarian cancer (stage III or IV according to the criteria of the International Federation of Gynecology and Obstetrics). One half of them were chemotherapy-naïve whereas the other half received CPT and PCT prior cytoreduction [32 (link)]. The patients were between 36 and 88 years old. The tumors were fixed in 4% formalin, embedded in paraffin, and cut into 3 μm sections. Deparaffinization, rehydration and epitope retrieval were performed using Envision Flex Target Retrieval Solution (Dako, Glostrup, Denmark). The cancerous nature of the tissues was identified using standard H + E staining by a pathomorphologist. Cytochemical detection of senescence-associated β-galactosidase (SA-β-Gal) was conducted following methodology described by Dimri et al. [33 (link)] and planimetric analyses of a green-stained area reflecting the presence of SA-β-Gal-positive cancer cells were performed as described in [10 (link)].

Paraffin-embedded samples from leiomyosarcomas were available from 57 patients. All cases were histologically and immunohistochemically validated. Immunohistochemistry for HDAC4 (1:100), MEF2C an SKP2 was performed by an automated immunostainer (Dako Autostainer). Antigen retrieval was performed with cytrate buffer at pH 6 for HDAC4 and at pH 9 with EnVision FLEX Target Retrieval Solution (Dako) for MEF2C and SKP2. All tumors were scored for the intensity of signal (range from 0 = no expression, to 4 = strong expression). Mean of intensity and percentage of duplicate cores were used for the final analysis.

Tissues were fixed in 4% buffered formalin, descaled by EDTA and embedded in paraffin. For immunohistochemical staining^{19 (link)}. Two micrometers of bone marrow tissue sections were incubated with EnVision Flex Target Retrieval Solution, pH low (K8005, DAKO) and stained with primary antibodies directed against SAMHD1 (12586-1-AP, Proteintech, 1:3000) and against CD34 (IR632, DAKO) for 40 min at room temperature. Polymeric secondary antibodies coupled to HRPO peroxidase and DAB were used for visualization (REAL EnVision Peroxidase/DAB +, K5007, DAKO). Tissue samples were analyzed by light microscopy after counterstaining with Meyer’s hematoxylin (K8008, DAKO). Two pathologists, who were blinded to clinical history and therapeutic response, independently scored the SAMHD1 IHCs. They evaluated all tissue sections for nuclear SAMHD1 staining using a four-stage staining score: 0 = negative; 1 = weak intensity of staining; 2 = strong intensity of staining in <25% of blasts; and 3 = strong intensity of staining in more than 25% of blasts. IHC staining scores of 0 and 1 were defined as ‘no or low expression’ and IHC staining scores of 2 and 3 were defined as ‘high SAMHD1 expression’. Membranous CD34 staining for the quantification of the number of AML blasts was evaluated using a two-stage staining score: 0 = negative; 1 = positive.

Materials were from Sigma Aldrich (St. Louis, Mo, USA) except rK39 immunochromatographic test strips (InBios International, Seattle, WA, USA), anti-human CD4 (clone 4B12, Novocastra, Newcastle upon Tyne, UK,), Chemokine Receptor 4 (CCR4, clone 1G1), CD3 Peridinin chlorophyll (PerCP, clone SK7), CD8 Alexa Fluor 488 (clone RPA-T8), CD8 PerCP (clone SK1), CCR4 Phycoerythrin (PE, clone 1G1, BD Biosciences, San Jose, CA, USA), PD-1(clone EH12.2H7) and CD127 (clone A019D5) APC (Biolegend, San Diego, CA, USA), IL-5 (clone 9906, R&D Systems, Minneapolis, MN, USA), CCL17 (clone N-20), IL-10 (clone E-10), PD-1 (clone C-20), Perforin H (clone H-315), Granzyme B (clone 2C5, Santa Cruz Biotechnology, Dallas, TX, USA), antibody diluent, Target Retrieval Solution Citrate pH 6 (S2369), secondary detection system EnVision™ G|2 System/AP-Rabbit/Mouse (Permanent Red), EnVision™ FLEX Target Retrieval Solution, EnVision™ DuoFLEX Doublestain System (Dako, Glostrup, Denmark), RNALater and RNAqueous kit (Ambion, Austin, TX, USA), One-step reverse transcription kit from Qiagen (Hilden, Germany) and Bio-Plex Pro™ Human Chemokine Panel 40-Plex (BioRad, Hercules, CA, USA).

Mice were immersion fixed in 4% PFA rather than perfused because of their small size, brains embedded in paraffin and 7 μm sections obtained as described (Deters et al., 2008 (link)). For antigen retrieval, the sections were microwaved in AR buffer (Dako Envision Flex Target Retrieval Solution low pH #K8005) for 15 min on low power before the buffer started to boil. The sections were then left to cool for 1h in the AR buffer at RT. Blocking was done in 20% FBS, 1% BSA in TBST for 1h at RT. Primary antibodies were used over night at 4°C and secondary antibodies for 1.5h at RT. Primary antibodies were Myc-Tag rabbit mAb (Cell Signaling Technologies, #71D10, used at 1:100) and anti-Human PHF-Tau Monoclonal Antibody AT8 (Thermo Fisher, MN1020, used at 1:400). Secondary antibodies were polyclonal goat anti-rabbit IgG biotinylated (Dako, #E0432, used at 1:500) and polyclonal rabbit anti-mouse IgG biotinylated (Dako, #E0413, used at 1:500). The VectaStain Elite ABC Kit #PK6102 was used and Envision Flex DAB chromogen (Dako, #DM827) and Envision Flex Substrate Buffer (Dako, # DM823) for DAB development. Haematoxylin was used for counter-staining.

Our previous work described the immunohistochemical stainings of CD3, CD8, Ki67, and cytokeratin used to determine the phenotypic subgroups [31 (link)].
The immunohistochemistry of the angiogenic growth factors VEGF, bFGF, and PDGF-bb was carried out using a similar method used to describe Ki67 and cytokeratin stainings [31 (link)]. First, slides were pretreated for 15 min with an EnVision Flex target retrieval solution (Dako, Santa Clara, CA, USA, DM828) at 98 °C in a pretreatment module (Agilent Technologies Inc., Dako, Santa Clara, CA, USA). Next, the pretreated slides were incubated with primary antibodies (mouse polyclonal anti-human VEGF (PharMingen, San Diego, CA, USA, diluted to 1:100), rabbit polyclonal anti-human bFGF (Bioss, Boston, Massachusetts, USA, diluted to 1:800), and rabbit polyclonal anti-human PDGF-bb (Thermo Scientific, Waltham, Massachusetts, USA, diluted to 1:500) in an Autostainer 480 (Lab Vision Corp, Fermont, CA, USA) overnight at room temperature. Subsequently, the slides were treated for 20 min with HRP-labeled EnVision Flex/HRP secondary antibodies (Dako, Santa Clara, CA, USA, SM802), which were then visualized by 10 min incubation with EnVision Flex DAB chromogen (Dako, Santa Clara, CA, USA, DM827). Finally, the slides were counterstained with Meyer’s hematoxylin and washed in tap water.

The FFPE sections were immunostained using the Dako EnVision™ Flex+ System (K8012; Dako, Glostrup, Denmark). Deparaffinization and epitope unmasking were carried out in a PT-Link using an EnVision™ Flex target retrieval solution (Dako, Carpinteria, CA, USA). The sections were treated with 0.3% hydrogen peroxide (H₂O₂) for 5 min to block endogenous peroxidase. Sections were incubated overnight at 4°C with the following antibodies: EYA2 (ab95875, Abcam), PTEN (ab31392, Abcam) and p-AKT (clone D9E, Cell Signaling Technology). The specimens were subsequently treated with EnVision™ Flex linker mouse or rabbit (15 min), EnVision™ Flex/HRP enzyme (30 min), and 3′3-diaminobenzidine tetrahydrochloride (10 min). The samples were counterstained with hematoxylin, dehydrated and mounted on a Richard-Allan Scientific Cyto seal XYL (Thermo Scientific, Waltham, MA, USA). The protein expression was scored semi-quantitatively based on the percentage of positive cells utilizing the following scale: +, <25%; ++, 25–49%; +++, 50–74%; and ++++, 75–100%.