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.
Immpress polymer detection system
Manufactured by Vector Laboratories
The ImmPRESS polymer detection system is a highly sensitive and versatile immunohistochemistry (IHC) detection reagent developed by Vector Laboratories. It utilizes a polymer-based approach to enhance the visualization of target antigens in tissue samples. The system is designed to provide consistent and reliable results in a wide range of IHC applications.
Automatically generated - may contain errors
Lab products found in correlation
Antigen retrieval solution, by Vector Laboratories (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)
Immpact novared, by Vector Laboratories (1 mentions)
Hematoxylin qs, by Vector Laboratories (1 mentions)
Bloxall endogenous peroxidase and alkaline phosphatase blocking solution, by Vector Laboratories (1 mentions)
Nimp r14, by Abcam (1 mentions)
3 protocols using immpress polymer detection system
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
Quantification of Lung Neutrophils
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