Immunohistochemistry was performed as described previously (Shukla et al., 2015 (link)). We used Novolink Polymer (Leica), as per the manufacturer’s instructions. Muscle sections prepared from human patient tissues were stained with SIRT1 antibody (Cell Signaling Technology) and NOX4 antibody (Abcam). Each muscle fiber in a cross-sectional field of view was given an intensity score by evaluating staining intensity of positive staining (0 = none, 1 = weak, 2 = intermediate, 3 = strong). The histoscore was calculated by multiplying the percentage of fibers (0–100) with the particular score by its corresponding intensity score (0–3). All the scores in a given section were added, and the value was then divided by 100 to attain a score between 1 and 3.
Novolink polymer
Manufactured by Leica camera
Sourced in United States
The Novolink Polymer is a laboratory equipment product designed for use in various scientific and research applications. It serves as a versatile reagent that can be used to enhance the visualization of target molecules during immunohistochemical or in situ hybridization procedures. The Novolink Polymer provides a streamlined and efficient method for signal amplification, allowing for clear and reliable detection of the desired targets.
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Lab products found in correlation
B220 antibody clone ra3 6b2, by BD (2 mentions)
Novocastra post primary, by Leica camera (2 mentions)
3 3 diaminobenzidine chromogen dab, by Leica camera (2 mentions)
24 pax 5, by BD (2 mentions)
Sirt1 antibody, by Cell Signaling Technology (1 mentions)
Nox4 antibody, by Abcam (1 mentions)
Bond intense r detection system, by Leica camera (1 mentions)
Bond wash solution, by Leica camera (1 mentions)
Cytoseal 60, by Thermo Fisher Scientific (1 mentions)
Bond dewax solution, by Leica camera (1 mentions)
Novolink polymer detection kit, by Leica camera (1 mentions)
Novolink polymer detection kit, by Leica Biosystems (1 mentions)
E330 digital camera, by Olympus (1 mentions)
Bh 2 microscope, by Olympus (1 mentions)
Rabbit anti nnos, by Cell Signaling Technology (1 mentions)
Leica dmi6000 microscope, by Leica camera (1 mentions)
C jun antibody, by Cell Signaling Technology (1 mentions)
Citrate buffer, by Merck Group (1 mentions)
Hematoxylin, by Leica camera (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
9 protocols using novolink polymer
1
Quantitative Immunohistochemistry Analysis
2
Immunohistochemical Profiling of ILC3 in Lung
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Immunohistochemical staining of human, NHP and mouse formalin-fixed paraffin-embedded (FFPE) lung sections were initially dewaxed in xylene prior to hydrating with decreasing graded alcohol and methanol passages. Antigen was retrieved via heat treatment in 92°C and EDTA buffer pH 8. Tissue staining with RORγt (Clone 6F3.1, Millipore for mouse; clone Q31–378, BD Bioscience for NHP and human), CD3 (clone SP7, Thermofisher for human, NHP and mouse) or PAX5 (Clone 24/Pax-5, BD Pharmingen, for human and NHP) or B220 antibody (clone RA3–6B2, BD Pharmingen) was performed for one hour in a humid chamber. Tissues were washed in Tris buffered saline pH7.4–7.6 prior to incubation with secondary antibody (Novocastra Post Primary, Leica) and polymer (Novolink Polymer, Leica). To develop the reaction, tissues were incubated with 3,3’-Diaminobenzidine chromogen (DAB, Leica). Singly stained sections (PAX5, B220) were incubated with DAB for 5 minutes and tissues receiving double staining (RORγt and CD3) were incubated overnight. Tissues were counterstained with haematoxylin and rinsed in water. All tissues were mounted with coverslips using glycerol mounting medium. CD3−RORγt+ ILC3 were quantified in the slides. Images were analyzed manually by counting the number of ILC3 cells per field. The analysis was done in a blinded manner.
3
Chromogenic IHC Staining of FFPE Tissue
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Chromogenic IHC was performed on formalin-fixed and paraffin-embedded (FFPE) tissue sections (5-µm thickness) using the Leica Bond III Autostainer system (Leica Biosystems, Deer Park, IL, USA) following the manufacturer’s instructions (25 (link)). The details of the primary antibodies used, their dilutions, and the procedure for antigen retrieval are summarized in Table 1 . Slides were deparaffinized in Bond Dewax solution (Leica, AR9222) and hydrated in Bond Wash solution (Leica, AR9590). Heat-induced antigen retrieval was performed at 100 ℃ in either Bond-Epitope Retrieval Solution 1 pH 6.0 (Leica, AR9961) or Bond-Epitope Retrieval Solution 2 pH 9.0 (Leica, AR9640). Antigen retrieval was followed by a 5-minute peroxide blocking step (IPB5000L, Biocare Medical, Pacheco, CA, USA), after which slides were incubated with the primary antibody followed by Leica Post Primary and Novolink Polymer (Leica, RE7260-CE) secondary reagents. Antibody detection with 3,3'-diaminobenzidine (DAB) and hematoxylin counterstain was performed using the Bond Intense R detection system (Leica, DS9263). Stained slides were dehydrated and coverslipped with Cytoseal 60 (23-244256, Fisher Scientific, Pittsburgh, PA, USA). Appropriate positive controls were used for each assay.
4
Immunohistochemical Profiling of ILC3 in Lung
5
Immunohistochemical Analysis of EpCAM and Ki67
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Serial tissue sections (4 µm) were H&E stained and analyzed by an expert who was blinded for the treatment groups. Immunohistochemical staining was performed on paraffin-embedded spheres and mouse tumor tissues using antibodies against the Epithelial Cell Adhesion Molecule (EpCAM) and Ki67 (Santa Cruz, CA, USA). Slides were dried, dewaxed in xylene and rehydrated using a decreasing alcohol series. After blocking of endogenous peroxidase with H2O2, antigen retrieval was performed in 10 mM citrate buffer, pH 6. Subsequently, slides were blocked with Protein Block (Novolink Polymer Detection Kit, RE7150-K, Leica). Primary antibodies were incubated at 4°C overnight, followed by Post Primary and Novolink™ Polymer (Novolink Polymer Detection Kit, RE7150-K, Leica). Staining was visualized using 3,3-diaminobenzidine (DAB), and nuclear counterstaining was performed using hematoxylin (Novolink Polymer Detection Kit, RE7150-K, Leica Biosystems, Germany). Slides were dehydrated and embedded in Histofluid (6900002; Marienfeld, Lauda Koenigshofen, Germany). Images were recorded at 40× to 400× magnification using an Olympus BH-2 microscope and an Olympus E330 digital camera. The staining intensity was classified into 0 (no staining), 1+ (weak), 2+ (moderate), 3+ (strong), and the average of positively stained cells was recorded.
6
Immunohistochemical Analysis of eNOS and nNOS in Mn-Treated Mice
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One day after MnCl2 treatment, corpora cavernosal tissues of both Mn-treated mice and their controls were simultaneously sampled to examine their immunohistochemical (IHC) staining. After formalin fixing, the cavernosal tissue section (5 μm) immersed in citrate buffer was applied to a heart-induced epitope retrieval for 15 minutes; it was next reacted with the primary antibody. Mouse anti-eNOS (1:200) (Cell Signaling Technology Inc, Danvers, MA, USA) binds to eNOS as a primary antibody, and rabbit anti-nNOS (1:500) (Cell Signaling Technology Inc). The slide was kept at room temperature for 60 minutes. The slide was washed with phosphate-buffered saline (PBS) for 10 minutes, reacted with the secondary antibody attached with biotin for 30 minutes, and then reacted with the chromogen 3,3′-diaminobenzidine tetrahydrochloride for 5 minutes. The slide was then reacted with NoVolink polymer (Leica, polymer detection system) for 30 minutes, and it was washed with PBS three times. The IHC staining was examined by one researcher.
7
Immunohistochemical Quantification of c-Jun
8
Immunohistochemical Analysis of Ki-67 Expression
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The slices of hippocampus were prepared as above and were put in boiling epitope retrieval solution (citrate buffer) (Sigma) for 10–15 min (based on kit instructions). Then, samples were washed 2-times with washing buffer solution (Sigma). Hydrogen peroxide solution was put on the slices for 15 min. Samples were washed again with washing buffer solution. Then, slices were incubated in anti-rat Ki-67 primary antibody (concentration: 1/100) (Abcam; ab 16667) at 37°C for 30 min, and again washed with washing buffer solution. Then, they were incubated in secondary antibody + washing buffer solution for 20 min at room temperature and were washed in washing solution and incubated in Novolink Polymer (Leica) + washing buffer solution for 10 min at room temperature and washed again. Chromogen solution substrate (DAB + substrate) was put on the samples for 15 min and washed in the washing solution. Then, they were washed with running water and stained with hematoxylin (detection kit: Leica).
9
Immunohistochemical Identification of B and T Cells
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After fixing the slides with cold acetone for 10 min, they were kept at -20 °C until used. In brief, cells on the glass slide were blocked for endogenous peroxidase and protein background by 3% (v/v) H 2 O 2 and 1% (w/v) bovine serum albumin (Merck Millipore, USA) in 0.25% phosphate-buffered saline-Triton X-100 (PBST), followed by incubation with the monoclonal mouse antihuman Pax5 antibody (1EW; Leica, USA) at a dilution of 1:50 at 4 °C for 12-14 h. For T-cell detection, cells were treated as B cells above except for using the ready-to-use monoclonal mouse anti-human CD3 antibody (LN10; Leica, USA). In both cases the signal of the bound antibody was amplified by Novolink polymer (Leica, USA) for 15 min and colour was developed with by 3,3'diaminobenzidine (DAB; Leica). Slides were counterstained with Mayer's haematoxylin for 1 min. Cytological smears from normal canine LN were used as negative control. Pax5 showed a nuclear staining in B cells while CD3 showed immunoreactivity in the cytoplasm of T cells. B-cell or T-cell lymphomas were identified if at least 60% of the cells revealed expression of Pax5 or CD3, respectively (Sirivisoot et al., 2017) .
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