Lung lobes were perfused with 10% formalin, fixed and paraffin embedded. For immunofluorescent staining, lung sections were cut, immersed in xylene to remove paraffin and then hydrated in 96% alcohol and phosphate-buffered saline. Antigens were unmasked with a DakoCytomation Target Retrieval Solution (Dako, Carpinteria, CA, USA), and non-specific binding was blocked with 5% (v/v) normal donkey serum and Fc block (BD Pharmingen). Endogenous biotin was neutralized by adding avidin, followed by incubation with biotin (Sigma Aldrich). Sections were probed with anti-B220 to detect B cells (clone RA3-6B2, BD Pharmingen; dilution: 1/100) and anti-CD3 to detect T cells (clone M-20, Santa Cruz Biotechnology, Santa Cruz, CA; dilution: 1/100). For analysis of B-cell follicles, follicles were outlined with the automated tool of the Zeiss Axioplan 2 microscope (Zeiss, Thornwood, NY, USA), and total area and average size was calculated in squared microns.
Dakocytomation target retrieval solution
Manufactured by Agilent Technologies
Sourced in United States
The DakoCytomation Target Retrieval Solution is a laboratory reagent used in immunohistochemistry and in situ hybridization procedures. It is designed to facilitate the antigen retrieval process, which is a crucial step in preparing tissue samples for analysis.
Automatically generated - may contain errors
Lab products found in correlation
Fc block, by BD (3 mentions)
Axioplan 2 microscope, by Zeiss (3 mentions)
Anti b220 clone ra3 6b2, by BD (2 mentions)
Clone ra3 6b2, by BD (1 mentions)
Clone m 20, by Santa Cruz Biotechnology (1 mentions)
Envision labeled peroxidase system, by Agilent Technologies (1 mentions)
Anti cd3, by Agilent Technologies (1 mentions)
Ci a3 1, by Novus Biologicals (1 mentions)
Anti tnf α, by Santa Cruz Biotechnology (1 mentions)
Anti bax, by Santa Cruz Biotechnology (1 mentions)
Anti cd3 clone m 20, by Santa Cruz Biotechnology (1 mentions)
E1l3n, by Cell Signaling Technology (1 mentions)
Protein block serum free, by Agilent Technologies (1 mentions)
Decloaking chamber, by Biocare Medical (1 mentions)
Biotinylated anti rabbit secondary antibody, by Vector Laboratories (1 mentions)
Avidin biotin complex, by Vector Laboratories (1 mentions)
Permount mounting media, by Thermo Fisher Scientific (1 mentions)
9 protocols using dakocytomation target retrieval solution
1
Quantifying B-cell Follicle Dynamics in Lung Tissue
2
IHC Staining for CD56 Expression
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4-μm-thick sections of formalin-fixed and paraffin-embedded blocks were prepared for IHC staining, using Envision Labeled Peroxidase System (DAKO, Carpentaria, CA, USA). After de-paraffinization and rehydration, the sections were washed with distilled water and then, antigen retrieval was performed by DAKO cytomation target retrieval solution (DAKO, Carpentaria, CA, USA) at pH=9, for 20 minutes. Then, the sections were incubated with anti-CD56 antibody (ready to use, Clone 1B6, Novocastra, Newcastle, UK) for 30 minutes. 3, 3 di-aminobenzidine (DAB liquid, DAKO Corporation, Denmark) was used as chromogen. Osteoblasts were used as internal positive control.(26 (link),27 (link)) Primary antibody was replaced by PBS solution in negative control sections. Brown staining in the cell membrane, cytoplasm or both in the epithelial component was considered as positive. Positive staining was considered “extensive”, when more than 50% of epithelial cells showed immunoreaction, and was considered “focal”, when 1-50% of epithelial cells were positive. Data were analyzed with SPSS 11, using chi-square test. P-value (PV) was approximated using Monte-Carlo method and was considered significant at P<0.05. Study groups with less than 10 cases were not considered in the statistical analysis.
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4-μm-thick sections of formalin-fixed and paraffin-embedded blocks were prepared for IHC staining, using Envision Labeled Peroxidase System (DAKO, Carpentaria, CA, USA). After de-paraffinization and rehydration, the sections were washed with distilled water and then, antigen retrieval was performed by DAKO cytomation target retrieval solution (DAKO, Carpentaria, CA, USA) at pH=9, for 20 minutes. Then, the sections were incubated with anti-CD56 antibody (ready to use, Clone 1B6, Novocastra, Newcastle, UK) for 30 minutes. 3, 3 di-aminobenzidine (DAB liquid, DAKO Corporation, Denmark) was used as chromogen. Osteoblasts were used as internal positive control.(26 (link),27 (link)) Primary antibody was replaced by PBS solution in negative control sections. Brown staining in the cell membrane, cytoplasm or both in the epithelial component was considered as positive. Positive staining was considered “extensive”, when more than 50% of epithelial cells showed immunoreaction, and was considered “focal”, when 1-50% of epithelial cells were positive. Data were analyzed with SPSS 11, using chi-square test. P-value (PV) was approximated using Monte-Carlo method and was considered significant at P<0.05. Study groups with less than 10 cases were not considered in the statistical analysis.
3
Lung Tissue Immunofluorescence Analysis
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Immunofluorescence staining was done as described before (2 (link)). FFPE lung sections were cut, immersed in xylene, and then hydrated in alcohol and PBS. Antigens were unmasked using DakoCytomation target retrieval solution (Dako), and non-specific binding was blocked by adding 5% (vol/vol) normal donkey serum and Fc block (BD). Avidin was used to neutralize endogenous biotin, followed by incubation with biotin (Sigma-Aldrich). Sections were then probed with anti-B220 (clone RA3-6B2, BD) to detect B cells. For analysis of B-cell follicles, follicles were outlined with an automated tool of the Zeiss Axioplan 2 microscope (Zeiss), and total area and average size were calculated in squared microns.
4
CD24 Immunohistochemistry Scoring Protocol
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After antigen retrieval in Dako Cytomation Target Retrieval Solution (DAKO, Carpinteria, CA, U.S.A.), slides were stained with primary antibodies (CD24, ML5 clone) and biotin-conjugated secondary antibodies, followed by incubation with Streptavidin Peroxidasee and Substrate-Chromogen solution (DAKO). Blinded immunohistochemical scoring was performed by a breast pathologist. CD24 staining was detected at the membrane and cytoplasm of tumor cells, and scoring was done as follows: 0, 0–10% of positive tumor cells; 1+, 10–25% of positive tumor cells; 2+, 25–50% of positive tumor cells; 3+, more than 50% of positive tumor cells. The cases were divided into negative/low, if scored 0 or 1+, and significantly positive cases, when scored as 3+. Patients initially presenting with metastases of any type, local or regional recurrence, or another primary cancer were excluded (n = 10) from the survival analysis.
5
Immunostaining of Lung Tissue Sections
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We microwaved lung sections in DakoCytomation Target Retrieval Solution (Dako) for 10 minutes at 100°C and restricted endogenous peroxidase activity using the method of Brown et al.30 We immunostained sections using a Histofine Simple Stain kit (Nichirei Corporation) with rabbit polyclonal anti‐CD3 (1:400; DakoCytomation), rabbit monoclonal anti‐PAX5 (1:50; D7H5X; Cell Signaling Technology), rat monoclonal anti‐F4/80 (1:50; CI‐A3‐1; Novus Biologicals), goat polyclonal anti‐TNF‐α (1:100; Santa Cruz Biotechnology), mouse monoclonal anti‐IL‐6 (1:100; Leica Biosystems Newcastle Ltd.), transforming growth factor‐β (TGF‐β, 1:100; Santa Cruz Biotechnology), anti‐BAX (1:100; Santa Cruz Biotechnology) or mouse monoclonal anti‐8‐hydroxydeoxyguanosine (8‐OHdG) antibody (5 µg/mL; JaICA; Nikken SEIL Co., Ltd.), as previously described.21 We counted the 8‐OHdG‐positive cell numbers per field of view in lungs from each group (n = 6) in a blinded manner. Further, we compared the means of the positive values between each group using an image analysis system (MacScoop version 2.5, Mitani).
6
Immunofluorescent Staining of FFPE Lung Sections
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For immunofluorescent staining, formalin-fixed and paraffin-embedded (FFPE) lung sections were cut, immersed in xylene, and then hydrated in 96% alcohol and PBS. Antigens were unmasked using DakoCytomation target retrieval solution (Dako), and nonspecific binding was blocked by adding 5% (vol/vol) normal donkey serum and Fc block (BD). Avidin was used to neutralize endogenous biotin, followed by incubation with biotin (Sigma-Aldrich). Sections were then probed with anti-B220 (clone RA3-6B2; BD) and anti-CD3 (clone M-20; Santa Cruz Biotechnology) to detect B and T cells, respectively. For analysis of B cell follicles, follicles were outlined with an automated tool of the Zeiss Axioplan 2 microscope (Zeiss), and total area and average size was calculated in squared microns.
7
PD-L1 Expression in FFPE Tissue
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4–5-μm formalin-fixed paraffin embedded tissue (FFPE) of human NPC tissue, A549 add C666-1 cells specimen were deparaffinized, rehydrated, and quenched with 1.5% H2O2. For antigen retrieval, slides were treated with Dako Cytomation Target Retrieval Solution (Dako, Carpinteria, CA) in a steam bath at 95 °C for 45 min. After equilibration in PBS for15 min, slides were placed in an auto stainer apparatus (Dako) and incubated with anti-PD-L1 antibody (E1L3N™, Cell Signaling Technology, Danvers, MA) at 1:200 dilution at room temperature for 30 min. Immunoreactivity was detected using the Dako EnVision method according to the manufacturer's instructions. For negative controls, slides were subjected to the same procedure, including antigen retrieval, except for omission of the primary antibody. The results were reviewed independently by 2 surgical pathologists, who were blinded to the clinical or pathological information of these patients. A semi-quantitative scale from 0 to 100% was used to grade (0~+++) of PD-L1 stained cancer cells and mesenchymal cells. The average score of replicate samples was used in the subsequent analyses.
8
Salmonella Immunodetection in Tissue Samples
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Slides were treated with DakoCytomation Target Retrieval Solution (Dako, Carpinteria, CA) in a Decloaking Chamber (Biocare Medical, Concord, CA) heated to 125°C and then cooled to 90°C for 10 sec, before cooling with the lid removed for 10 min to unmask epitopes for Salmonella detection. Slides were then transferred to a Dako Universal Training Center automatic immunostainer for all subsequent steps at RT. Endogenous peroxidase was inhibited in 3% H2O2 for 5 min, followed by serum-free protein block (Dako) for 10 min. Sections were incubated with rabbit polyclonal anti-Salmonella antibody (1∶100) for 30 min (Novus Biologicals), followed by a biotinylated anti-rabbit secondary antibody (1∶200, Vector Laboratories, Burlingame, CA) for 30 min, and lastly avidin-biotin complex (Vector Laboratories) for 30 min. Signal was developed with 3,3′ diaminobenzidine tetrahydrochloride, counterstained with hematoxylin, and coverslipped prior to viewing with a light microscope.
9
Immunohistochemical Analysis of Extracellular Matrix Regulators
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