To evaluate tumor burden lung tissues were harvested, fixed in formaldehyde 10% and paraffin embedded following standard procedure. Consecutive sections every 200 µm were dewaxed and rehydrated and stained with the H&E (Bio-Optica, Milano Spa). The area of tumor nodules was quantified over consecutive sections and averaged (three sections/sample). Measurements and automatic thresholding were performed using Image J. The area occupied by tumor nodules was expressed as a function of the total lung lobe area. For CD8+ T cells infiltration and HA expression, sections were treated with antigen-retrieval solution (Vector laboratories) for 20 min at 120 °C. Slides were treated for 2 min in H2O2 to block endogenous alkaline phosphatase. After blocking in 10% goat serum in 0.1% Tween20 for 30 min slides were incubated overnight in a humidified chamber in a 1:500 dilution of anti-mouse CD8 (Invitrogen cat 14–0808–82) in blocking buffer or anti-HA 1:50 (Roche). Detection was performed using the ImmPRESS polymer detection system (Vector Laboratories), according to manufacturer’s Instructions. Ilastik software was used to automatic detect and segmentate CD8+ cells. Segmented images were further automatic analyzed for the number of foci using Image J software. The number of tumor-infiltrating CD8+ T cells was normalized on tumor nodule area.
Antigen retrieval solution
Manufactured by Vector Laboratories
Sourced in United States
Antigen retrieval solution is a laboratory reagent used to enhance the detection of target proteins in fixed tissue samples. It functions by breaking down and exposing the target antigens, which can be obscured by the fixation process, to allow for improved binding and visualization of specific antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Immpress polymer detection system, by Vector Laboratories (2 mentions)
Normal tissue microarray, by Novus Biologicals (2 mentions)
Abc kit, by Vector Laboratories (2 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (2 mentions)
Anti ha, by Roche (1 mentions)
Anti mouse ly6g 1a8, by BD (1 mentions)
Anti mouse ki67 d3b5, by Cell Signaling Technology (1 mentions)
Aperio imagescope, by Leica (1 mentions)
Methyl green, by Vector Laboratories (1 mentions)
Alexa 594, by Thermo Fisher Scientific (1 mentions)
Immpact dab diluent, by Vector Laboratories (1 mentions)
Axiovert s100, by Zeiss (1 mentions)
α sma, by Vector Laboratories (1 mentions)
Citrisolv, by Thermo Fisher Scientific (1 mentions)
α sma, by Merck Group (1 mentions)
Hematoxylin, by Vector Laboratories (1 mentions)
Mirneasy isolation kit, by Qiagen (1 mentions)
Mouse monoclonal antibody against beta actin, by Merck Group (1 mentions)
Normal donkey serum (nds), by Jackson ImmunoResearch (1 mentions)
Irdye 800cw secondary antibody, by LI COR (1 mentions)
20 protocols using antigen retrieval solution
1
Quantification of Tumor Burden and CD8+ T Cell Infiltration
2
Quantifying Tumor Burden and Cell Proliferation
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To assess tumor burden, lung tissues were harvested and fixed in formaldehyde 10% and paraffine embedded following standard procedure. Consecutive sections of 8 μm were dewaxed and rehydrated and stained with the H&E using (Bio-Optica, Milano Spa). The area of tumor nodules was quantified manually over consecutive sections and averaged (three sections/sample).
To identify neutrophils or proliferating cells within nodules, sections were treated with antigen-retrieval solution (Vector laboratories) for 20 min at 120°C. Slides were treated for 10 min in H2O2 and after blocking in 10% goat serum in 0.1% Tween 20 for 30 min and incubated overnight at 4°C with specific antibody diluted in PBS 0.1%Tween 20: anti-mouse Ly6G (1A8, BD Pharmingen, cat. no. 551459), or anti-mouse Ki67 (D3B5, Cell Signaling, cat. no. 12202s). Detection was performed using the ImmPRESS polymer detection system (Vector Laboratories), according to manufacturer’s Instructions. Automatic thresholding and measurements were performed using Ilastik or ImageJ software, respectively. Images were acquired by Leica microscope. For tumor burden, neutrophil and proliferating cells measurements slides were scored blindly by two independent operators.
To identify neutrophils or proliferating cells within nodules, sections were treated with antigen-retrieval solution (Vector laboratories) for 20 min at 120°C. Slides were treated for 10 min in H2O2 and after blocking in 10% goat serum in 0.1% Tween 20 for 30 min and incubated overnight at 4°C with specific antibody diluted in PBS 0.1%Tween 20: anti-mouse Ly6G (1A8, BD Pharmingen, cat. no. 551459), or anti-mouse Ki67 (D3B5, Cell Signaling, cat. no. 12202s). Detection was performed using the ImmPRESS polymer detection system (Vector Laboratories), according to manufacturer’s Instructions. Automatic thresholding and measurements were performed using Ilastik or ImageJ software, respectively. Images were acquired by Leica microscope. For tumor burden, neutrophil and proliferating cells measurements slides were scored blindly by two independent operators.
3
Anti-CNGB3 Antibody Generation and Characterization
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A peptide sequence of human CNGB3, KENEDKGKENEDKDKGREPEEKP-C, was synthesized by GenScript. A cysteine was added at the carboxy terminus for conjugation with keyhole limpet hemocyanin (Immunopure KLH, Pierce) using a SMPB cross-linker (Pierce) according to the manufacturer’s protocol. Female Balb/c mice were immunized with this conjugate, and spleens were isolated from the immunized mice. Lymphocytes released from the spleen were fused to mouse myeloma P3X cells, and hybridoma clones were screened by immunostaining as Ishikawa (positive) and A431 (negative) cells. A hybridoma clone was subcloned by a limited dilution, establishing the 3B2 clone. Paraffin tissue sections endometriosis patients (n=35) were obtained from Folio Biosciences (Columbus, OH). A normal uterine endometrium tissue microarray (60 cores) and a normal tissue microarray (60 cores) were obtained from Imgenex (San Diego, CA). Antigen retrieval was performed by autoclaving sections at 110 °C for 1 min in antigen retrieval solution (Vector Laboratories). Immunohistochemistry with 3B2 (mouse IgG) was performed using an ABC kit (Vector Laboratories) with a DAB colour reaction and hematoxylin counterstaining.
4
Histological Analysis of Neurodegeneration in Mice
5
Immunofluorescent Analysis of Lung Tissue
6
Immunohistochemical Analysis of Tumor Markers
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Immunohistochemical analysis was performed as described previously [26 (link)]. Tumor tissue sections were incubated in xylene and passed through series of graded alcohols followed by antigen retrieval using the antigen retrieval solution (Vector Labs, Burlingame, CA). Tissue sections were incubated in 3% H2O2 solution for 20 min and then subjected to blocking using the Vector Lab Blocking Kit. Tissue sections were incubated overnight with primary antibodies of Ki-67 (1:100), Cox-2 (1:50), and IL-1 beta (1:100) and then with secondary antibodies. Immunoreactivity was visualized by using the DAB substrate and counterstained with hematoxylin (Vector Lab, Inc.). The proliferative index was calculated as percentage of Ki-67-positive cells in five randomly selected microscopic fields. TUNEL analysis was performed using the In situ Cell Death Detection Kit according to manufacturer's protocol and DAPI was used to visualize the nuclei. Percentage of apoptotic cells were calculated from five randomly selected microscopic fields in each group.
7
Quantification of Protein Expression in Cells
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The miRNeasy isolation kit and the QuantiNova SYBR Green PCR mix were purchased from Qiagen (Valencia, CA). Running buffer, transfer buffer, NuPage 4–12% Bis-Tris gels, and Invitrolon polyvinylidene fluoride (PVDF) membranes (0.45 µm) were procured from Invitrogen (Carlsbad, CA) while the blocking buffer was from Li-Cor Biosciences (Lincoln, NE). Antigen retrieval solution and 4',6-diamidino-2-phenylindole (DAPI) were from Vector Laboratories (Burlingame, CA), and the normal donkey serum was purchased from Jackson ImmunoResearch Laboratories (Westgrove, PA). The following primary antibodies were used for immunohistochemistry and western blotting: rabbit monoclonal antibody against aquaporin 5 (1:100 or 1:5,000; Abcam Inc., Cambridge, MA); mouse monoclonal antibody against E-cadherin (1:100 or 1:5,000; BD Biosciences, San Jose, CA), rabbit polyclonal antibody against α-SMA (1:100 or 1:5,000; Abcam Inc.), and mouse monoclonal antibody against beta actin (1:10,000; Sigma-Aldrich Co., St. Louis, MO). Fluorescein isothiocyanate (FITC) or tetramethylrhodamine (TRITC) conjugated secondary antibodies (1:100, Jackson ImmunoResearch Laboratories) were used for immunohistochemical staining, while IRDye 680RD or IRDye 800CW secondary antibodies (1:5,000; Li-Cor Biosciences) were used for western blotting.
8
Immunofluorescence Staining of Paraffin-Embedded and Frozen Tissue
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Sections of paraffin embedded brains were dewaxed in Histoclear™ or Xylene (2 × 5 min). Slides were rehydrated through decreasing ethanol series (2 × 5 min: 100%, 3 min: 95%, 75%, 50%, 30%), washed in water (5 min) and PBS (5 min). Frozen sections were warmed to room temperature and washed in PBS (5 min) to remove OCT. If required, antigen retrieval was carried out by placing sections in antigen retrieval solution (Vector Laboratories) and microwaved until boiling for 20 min, after which they were allowed to cool (20 min). After antigen retrieval, slides were briefly dried, sections circumscribed with ImmEdge PAP pen (Vector laboratories Ltd, UK) and incubated in blocking buffer at room temperature (1 h) followed by incubation with primary antibodies (see Supplementary Material, Table S2 for list of primary antibodies) overnight at 4°C. Slides were washed in PBS with Tween-20 0.1% (v/v) (PBST, 4 × 10 min). Appropriate secondary antibodies (Supplementary Material, Table S2 ) were used at a dilution of 1:1000 in blocking buffer and incubated for 1 h at RT. In all cases 4′,6-Diamidino-2-Phenylindole (DAPI) was also added to secondary antibody mixture (1 μg/ml). Slides were washed in PBST (2 × 10 min) followed by PBS (2 × 10 min) and coverslips mounted with Mowiol (Polysciences Inc., Germany).
9
Monoclonal Antibody Development for Endometriosis
10
Immunohistochemical Analysis of Brain Samples
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Immunohistochemical studies were performed as described previously25 (link). Coronal brain sections were incubated in xylene and passed through series of graded alcohols and then subjected to antigen retrieval using the antigen retrieval solution (Vector Lab, Inc. CA). Tissue sections were incubated in 3% H2O2 solution for 20 min and then subjected to blocking using the vector lab blocking kit. Tissue sections were incubated overnight with Ki-67 (1:100), Cleaved Caspase-3 (1:200) and Bcl-2 (1:50) and then with secondary antibodies for 45 min at room temperature. Immunoreactivity was visualized by using the DAB substrate and counterstained with haematoxylin (Vector Lab, Inc. CA). The proliferative index was calculated as percentage of Ki-67-positive cells in five randomly selected microscopic fields at 20X per slide. TUNEL analysis was performed using the In situ Cell Death Detection Kit (Roche, Indianapolis, IN) as per the manufacturer’s protocol, and five randomly selected microscopic fields in each group were used to calculate the relative ratio of TUNEL-positive cells. DAPI was used to visualize the nuclei.
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