FFPE tumor samples from xenografts or primary patient samples were deparaffinized, then antigen retrieval was carried out using Diva Decloaker in a Biocare Medical decloaking chamber (120oC for 30 sec.) and slides were allowed to cool to room temperature. Slides were blocked with 0.2% BSA for 1 hour at room temperature. Primary and secondary antibodies (Supplemental Table 6 ) were incubated in 0.2% BSA for 1 hour at room temperature. When both goat and donkey derived antibodies were used, donkey-anti-goat secondary was applied first, washed 3×10 min. with PBS, then additional secondaries were added to prevent cross-species reactivity. DAPI was included during secondary antibody incubations to mark nuclei at 1 ug/mL. Coverslips were mounted with Prolong Gold and allowed to cure overnight at room temperature in the dark, then slides were stored at 4oC until imaging. Slides were imaged on a Leica SPE confocal at 40X magnification or scanned on an Olympus slide scanner at 10X and 20X magnification.
Medical decloaking chamber
Manufactured by Biocare Medical
Sourced in United States
The Medical Decloaking Chamber is a laboratory equipment designed to create a controlled environment for processing and preparing samples. It provides a secure and isolated space for various applications that require a specific atmosphere or conditions.
Automatically generated - may contain errors
Lab products found in correlation
Slide scanner, by Olympus (2 mentions)
Goat serum, by Merck Group (2 mentions)
High ph antigen retrieval solution, by Agilent Technologies (2 mentions)
3 3 diaminobenzidine dab chromogen, by Agilent Technologies (2 mentions)
Vector trueview, by Vector Laboratories (1 mentions)
Citrate based antigen unmasking solution, by Vector Laboratories (1 mentions)
Cd163, by Proteintech (1 mentions)
P ikba, by Cell Signaling Technology (1 mentions)
Anti rabbit alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Anti mouse alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Pd l1, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Autostainer xl, by Leica (1 mentions)
D2 40, by Cedarlane (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Labtec 2 chamber slides, by Thermo Fisher Scientific (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
8 protocols using medical decloaking chamber
1
FFPE Immunofluorescence Staining Protocol
2
Immunohistochemical Liver Slice Protocol
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Slide-mounted liver slices were washed twice in xylene for 3 minutes followed by washes in 100%, 95%, 70% and 50% ethanol for 3 minutes each. Slides were then washed with DI water and heated to 90C for 30 minutes in 1% citrate-based antigen unmasking solution (Vector Laboratories) using a Biocare Medical Decloaking Chamber. Slides were washed with TBS-0.025% Tween (TBST) and then blocked for 4 hours in TBST containing 1.5% BSA and 15% goat serum (Sigma Aldrich). Slides were incubated in primary antibodies at 4C overnight. Following primary antibody staining, slides were washed with TBST and incubated with secondary antibodies and DAPI (1:3,000) for 1 hour at room temperature. Slides were washed with TBST and autofluorescence quenched using Vector TrueView (Vector Labs). Fluoromount G mounting media was used to preserve fluorescence signal. Primary antibodies were used at the following concentrations: PyHsp70 1:1,000, PyCSP-488 1:500, p-p44/42 1:200 (Cell Signaling 4370), p-IK-Ba 1:200 (Cell Signaling 2850), p-Akt 1:100 (Cell Signaling 9271), CD163 1:500 (Proteintech 16646-1-AP), CLEC4F-647 1:100 (BioLegend 156804), PD-L1 1:200 (Cell Signaling 64988), B7H3 1:200 (Novus Bio NB600-1441). Secondary antibodies anti-mouse AlexaFluor-488, anti-rabbit AlexaFluor-594, and anti-rabbit AlexaFluor-647 (Invitrogen) were used at a 1:1,000 dilution.
3
Immunohistochemical Analysis of Galectin-1
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Formalin fixed paraffin embedded tissue was sectioned at 4-microns and dried overnight at 58°C. Slides were deparaffinized and hydrated stepwise followed by heat-induced epitope retrieval (HIER) in high pH antigen retrieval solution (Dako) at 110°C for 20 minutes using a Biocare Medical Decloaking Chamber. Slides were incubated in Galectin-1 antibody (R&D AF-1152) diluted at 1:50 for one hour at room temperature followed by incubation with polymer bound anti-goat secondary for 30 minutes (Vector Laboratories Immpress kit). Staining was visualized by incubation with 3, 3′-diaminobenzidine (DAB) chromogen (Dako, CA) for 5 minutes and slides were lightly counterstained with hematoxylin.
4
Immunohistochemical Staining of Lymphatics
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Sections were deparaffinised and rehydrated using the Leica Autostainer XL. Antigen retrieval was performed by heating slides in citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) using a Biocare Medical Decloaking Chamber (Biocare Medical, LLC, Concord, California, USA). Sections were washed three times in 1× phosphate-buffered saline (PBS, pH 7.4) for 5 min each, followed by incubation in blocking solution for 1 hour with 2% goat serum (Sigma-Aldrich, Missouri, USA) and 0.3% Triton X-100 (Sigma-Aldrich, Missouri, USA) in 1× PBS. Sections were then incubated in blocking solution with primary antibodies D2-40 (1:100, mouse monoclonal, Cedarlane Laboratories) and CD31 (1:20, rabbit polyclonal, Abcam, Cambridge, UK) overnight at 4°C. CD31 was used as a marker of blood vessel endothelial cells.18 (link) After three 10 min 1× PBS washes, sections were incubated with goat antimouse Alexa Fluor-555 (1:100; Thermo Fisher Scientific, Ontario, Canada) and goat antirabbit Alexa Fluor-647 (1:100; Thermo Fisher Scientific) secondary antibodies in blocking solution for 1 hour in the dark. After three washes in 1× PBS for 10 min each, the sections were covered with a coverslip (#1.5, Thermo Fisher Scientific) and aqueous mounting medium (Dako, Agilent Technologies, California, USA). All sections were treated at room temperature with mild agitation unless otherwise noted.
5
Immunohistochemical Analysis of Galectin-1 in TNBC
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Six cases of triple negative breast cancer from formalin fixed surgical archives of the University of Kentucky were selected and 4-micron thick sections were cut from each and dried overnight at 58°C. Slides were deparaffinized and hydrated stepwise followed by heat-induced epitope retrieval (HIER) in high pH antigen retrieval solution (Dako) at 110°C for 20 minutes using a Biocare Medical Decloaking Chamber. Slides were incubated in Galectin-1 antibody (Sigma #HPA000646) diluted at 1:400 for 30 minutes at room temperature followed by incubation with polymer bound anti-rabbit secondary for 30 minutes (Dako Envision+). Staining was visualized by incubation with 3, 3′-diaminobenzidine (DAB) chromogen (Dako, CA) for 3 minutes and slides were lightly counterstained with hematoxylin.
6
Immunohistochemical Staining of Prostate Cancer Tissue and Cell Lines
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For IHC deparaffinized formalin fixed and paraffin embedded PCa tissue and fixed PCa cell lines grown on Lab-TECII chamber slides (154526, Nalge Nunc international, Naperville IL 60563–1796, USA) were used. Duplicate PCa tissue slides were obtained from the pathology department of patients with confirmed PCa as defined by a trained pathologist. The protocol used for IHC was previously described in [21 (link)]. It only diverted for the deparaffination step. In short, to damask the cells and tissue they were heated with TEG buffer (Tris 6,06g, EGTA 0,959 in 5L, pH 8,5) in a pressure cocker (Biocare medical decloaking chamber, Concord, Ca, USA). The slides were then blocked with 1% (v/v) H2O2 in MeOH for 30 minutes and with 5% BSA (w/v) in horse serum (20% v/v) (ImmPRESS, Vector Laboratories, Burlingame, CA) and Tris buffered saline (80% v/v) before application of antibody. The LHCGR OASG04237 antibody (ProMab biotechnologies Inc. Catalog # 30751) was diluted 1:7500 for the tissue and 1:200 for the cell lines and left overnight at 4°C followed by 1 h at room temperature. Anti-mouse secondary antibody (ImmPRESS, MP-7402) was applied for 30 minutes at room temperature and development was achieved with AEC (3-amino-9-cabazole, ImmPRESS, SK4205). Omission of primary antibody was used as control on sections of prostate, testis tissue and the cell lines.
7
Immunohistochemical Analysis of AMPK Subunits in TNBC
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TNBC whole tissue samples were selected by the Markey Cancer Center Biospecimen Core. Four micrometer slides were deparaffinized and hydrated stepwise. Antigen retrieval was carried out in a Biocare Medical decloaking chamber at 95 °C for 20 min, followed by quenching of endogenous peroxidase activity and incubation with primary antibody overnight at 4 °C. The slides were subsequently incubated with Vector Laboratories ImmPRESS® anti-rabbit HRP polymer for 30 min at room temperature and staining was visualized with DAB (Dako). Antibody specific conditions were as follows: (1) AMPKα1: Dako high pH antigen retrieval buffer; 10-min DAB incubation; 1:175 dilution and (2) AMPKα2: Dako low pH antigen retrieval buffer; 5 min DAB incubation; 1:300 dilution. A pathologist who was blinded to the stage of disease scored the samples for staining intensity and distribution percentage on scales from 0 to 3. For staining intensity: 0 = negative; 1 = weak; 2 = moderate; and 3 = strong. For distribution percentage: 0 = 0%; 1 = 1%–10%; 2 = 11%–50%; and 3 = 51%–100%.
TNBC patient-derived xenografts (PDXs) were from Dr. Kathleen O’Connor’s laboratory and were immunostained as described above except the AMPKα1 dilution was 1:100 and the AMPKα2 dilution was 1:200.
TNBC patient-derived xenografts (PDXs) were from Dr. Kathleen O’Connor’s laboratory and were immunostained as described above except the AMPKα1 dilution was 1:100 and the AMPKα2 dilution was 1:200.
8
Immunofluorescence Staining of FFPE Samples
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FFPE tumor samples from xenografts or primary patient samples were deparaffinized, then antigen retrieval was carried out using Diva Decloaker in a Biocare Medical decloaking chamber (120°C for 30 sec.) and slides were allowed to cool to room temperature. Slides were blocked with 0.2% BSA for 1 hour at room temperature. Primary and secondary antibodies (Supplemental Table 1) were incubated in 0.2% BSA for 1 hour at room temperature. When both goat and donkey derived antibodies were used, donkey-anti-goat secondary (RRID:AB_2535853) was applied first, washed 3×10 min. with PBS, then additional secondaries were added to prevent cross-species reactivity. DAPI was included during secondary antibody incubations to mark nuclei at 1 ug/mL. Coverslips were mounted with Prolong Gold and allowed to cure overnight at room temperature in the dark, then slides were stored at 4°C until imaging. Slides were imaged on a Leica SPE confocal at 40X magnification or scanned on an Olympus slide scanner at 10X and 20X magnification.
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