Dab sparkle enhancer, by Biocare Medical - Product details

1

Immunohistochemical Analysis of Tissue Samples

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Tissue specimens were fixed in neutral formalin buffered saline (10%)
and embedded in paraffin. Hematoxylin and eosin staining was performed using
standard methods and tissue specimens from experimental animals were reviewed in
a blinded fashion by a clinical pathologist (M. M. I.) For Ki67 staining,
3–4 micron tissue sections were cut from paraffin blocks and baked
overnight in a dry slide incubator, then deparaffinized on a Shandon-Lipshaw
Varistain using a series of incubations in xylene, ethanol, then water. Antigen
retrieval was achieved by incubating slides in Tris-HCL 9.0 AR buffer in a T-FAL
OPTIMA pressure cooker. Slides were rinsed, then endogenous peroxidase was
blocked by immersing slides in 3% hydrogen peroxide for 5 minutes. Following
washes in nanopure water and TBS-20, primary antibody was applied at a dilution
of 1:200 (Ki67, MIB-1 Clone, Dako). Following primary antibody incubation,
slides were washed and then incubated with envision-labelled polymer-HRP
Anti-Mouse (Dako). Slides were washed, then DAB+ solution (DakoCytomation) was
added for a 15-minute incubation, after which slides were rinsed with nanopure
water. Chromogen signal was enhanced using DAB Sparkle Enhancer (Biocare).
Slides were washed, then counterstained with Harris Hematoxylin, dehydrated,
cleared, then mounted using Cytoseal (VWR).

Bader D.A., Hartig S.M., Putluri V., Foley C., Hamilton M.P., Smith E.A., Saha P.K., Panigrahi A., Walker C., Zong L., Martini-Stoica H., Chen R., Rajapakshe K., Coarfa C., Sreekumar A., Mitsiades N., Bankson J.A., Ittmann M.M., O’Malley B.W., Putluri N, & McGuire S.E. (2018). Mitochondrial pyruvate import is a metabolic vulnerability in androgen receptor-driven prostate cancer. Nature metabolism, 1(1), 70-85.

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2

Immunohistochemical Evaluation of PTEN Expression

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PTEN expression was assessed by immunohistochemistry (IHC) on freshly cut 4μm tissue sections of FFPE specimens that were deparaffinized, followed by heat-mediated antigen retrieval using 0.1M Tris-HCL (pH 9.0) buffer. To decrease nonspecific binding, sections were first treated with 3% hydrogen peroxide solution. Tissues were then incubated for 1 hour at room temperature with rabbit polyclonal PTEN (D4.3) antibody (Cell Signaling, Beverly, MA) at a 1:100 dilution in SignalStain antibody diluents (Cell Signaling), followed by incubation with Envision Labelled Polymer-HRP Anti-Rabbit (Dako, Carpenteria, CA), DAB+ solution (Dako), and DAB Sparkle Enhancer (Biocare, Concord, CA) for detection. Immunohistochemical results were evaluated independently by two pathologists (A.C and S.H.) who were blinded to the results of genetic analysis and patient outcome. As a specimen’s internal control, stromal cells were assessed for positive staining; samples devoid of staining in the internal controls were considered uninterpretable. Each section was scored for percent positivity (0–100%) and level of intensity (0–3). The H-score of each sample was calculated by multiplying the percentage and intensity scores.

Rimawi M.F., De Angelis C., Contreras A., Pareja F., Geyer F.C., Burke K.A., Herrera S., Wang T., Mayer I.A., Forero A., Nanda R., Goetz M.P., Chang J.C., Krop I.E., Wolff A.C., Pavlick A.C., Fuqua S.A., Gutierrez C., Hilsenbeck S.G., Li M.M., Weigelt B., Reis-Filho J.S., Osborne C.K, & Schiff R. (2017). Low PTEN Levels and PIK3CA Mutations Predict Resistance to Neoadjuvant Lapatinib and Trastuzumab without Chemotherapy in Patients with HER2 Over-Expressing Breast Cancer. Breast cancer research and treatment, 167(3), 731-740.

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3

Immunohistochemical Analysis of Tissue Samples

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Tissue specimens were fixed in neutral formalin buffered saline (10%)
and embedded in paraffin. Hematoxylin and eosin staining was performed using
standard methods and tissue specimens from experimental animals were reviewed in
a blinded fashion by a clinical pathologist (M. M. I.) For Ki67 staining,
3–4 micron tissue sections were cut from paraffin blocks and baked
overnight in a dry slide incubator, then deparaffinized on a Shandon-Lipshaw
Varistain using a series of incubations in xylene, ethanol, then water. Antigen
retrieval was achieved by incubating slides in Tris-HCL 9.0 AR buffer in a T-FAL
OPTIMA pressure cooker. Slides were rinsed, then endogenous peroxidase was
blocked by immersing slides in 3% hydrogen peroxide for 5 minutes. Following
washes in nanopure water and TBS-20, primary antibody was applied at a dilution
of 1:200 (Ki67, MIB-1 Clone, Dako). Following primary antibody incubation,
slides were washed and then incubated with envision-labelled polymer-HRP
Anti-Mouse (Dako). Slides were washed, then DAB+ solution (DakoCytomation) was
added for a 15-minute incubation, after which slides were rinsed with nanopure
water. Chromogen signal was enhanced using DAB Sparkle Enhancer (Biocare).
Slides were washed, then counterstained with Harris Hematoxylin, dehydrated,
cleared, then mounted using Cytoseal (VWR).

Bader D.A., Hartig S.M., Putluri V., Foley C., Hamilton M.P., Smith E.A., Saha P.K., Panigrahi A., Walker C., Zong L., Martini-Stoica H., Chen R., Rajapakshe K., Coarfa C., Sreekumar A., Mitsiades N., Bankson J.A., Ittmann M.M., O’Malley B.W., Putluri N, & McGuire S.E. (2018). Mitochondrial pyruvate import is a metabolic vulnerability in androgen receptor-driven prostate cancer. Nature metabolism, 1(1), 70-85.

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4

Quantifying Tumor-Infiltrating CD8+ Cells

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Tumor chunks were fixed in 10% formalin overnight at 4°C overnight, and subsequently transferred into 70% ethanol, embedded in paraffin, and sectioned at regular intervals. Slides were deparaffinized and hydrated using xylene, graded ethyl alcohol, and dH2O. After antigen retrieval, 15 minutes of steaming at 90°C with pressure in 0.1M Tris-HCl, pH 9.0, sections were treated with 3% hydrogen peroxide solution for 5 minutes. Sections were incubated with primary antibody (CD8a, Cell Signaling 98941 diluted 1:100) for 1hr at RT. Sections were then incubated with Envision Labelled Polymer-HRP (Dako) for 30 minutes at RT. DAB+ solution (DakoCytomation) was then added to section, incubated for 15 minutes, followed by application of DAB Sparkle Enhancer (Biocare). Sections were counterstained with Harris Hematoxylin. Counting of CD8a+ stained cells was done using the Count Tool in Adobe Photoshop.

Bowling E.A., Wang J.H., Gong F., Wu W., Neill N.J., Kim I.S., Tyagi S., Orellana M., Kurley S.J., Dominguez-Vidaña R., Chung H.C., Hsu T.Y., Dubrulle J., Saltzman A.B., Li H., Meena J.K., Canlas G.M., Chamakuri S., Singh S., Simon L.M., Olson C.M., Dobrolecki L.E., Lewis M.T., Zhang B., Golding I., Rosen J.M., Young D.W., Malovannaya A., Stossi F., Miles G., Ellis M.J., Yu L., Buonamici S., Lin C.Y., Karlin K.L., Zhang X.H, & Westbrook T.F. (2021). Spliceosome-Targeted Therapies Trigger an Antiviral Immune Response in Triple-Negative Breast Cancer. Cell, 184(2), 384-403.e21.

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5

Quantifying p-HER2 and ER+ in Xenograft Tissues

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IHC staining was executed with the help of the Pathology Core at the Lester and Sue Smith Breast Center, Baylor College of Medicine. Tissue sections were incubated at 58°C overnight in a dry slide incubator and deparaffinized in xylene and graded alcohol washes. Antigen retrieval was performed in 0.1 M Tris-HCl pH 9.0 (p-HER2 and ERα), followed by quenching in 3% H₂O₂. The following antibodies were used to stain for 1 hour at room temperature: ERα (clone 6F11, Novocastra, 1:200) and p-HER2 (Cell Signaling, 2243L, 1:50). After washing in TBS, EnVision labeled polymer-HRP anti-mouse or anti-rabbit antibodies (Dako) were added for 30 minutes at room temperature. Slides were washed with TBS and then developed with DAB⁺ solution (Dako) and DAB sparkle enhancer (Biocare). After washing in TBS, slides were counterstained with hematoxylin, dehydrated, and cleared before coverslipping with Cytoseal (VWR). p-HER2 and ER⁺ stained cells were quantified in lung/ovary sections of L755S fat pad (FP) xenografts and ovary sections from HER2 WT and HER2 S310F or L755S MIND xenograft mice.

Kalra R., Chen C.H., Wang J., Salam A.B., Dobrolecki L.E., Lewis A., Sallas C., Yates C.C., Gutierrez C., Karanam B., Anurag M., Lim B., Ellis M.J, & Kavuri S.M. (2022). Poziotinib Inhibits HER2-Mutant–Driven Therapeutic Resistance and Multiorgan Metastasis in Breast Cancer. Cancer Research, 82(16), 2928-2939.

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Dab sparkle enhancer

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5 protocols using dab sparkle enhancer

Immunohistochemical Analysis of Tissue Samples

Immunohistochemical Evaluation of PTEN Expression

Immunohistochemical Analysis of Tissue Samples

Quantifying Tumor-Infiltrating CD8+ Cells

Quantifying p-HER2 and ER+ in Xenograft Tissues

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