We performed IHC in paraffin-embedded samples obtained from the CGGA sample bank. IHC analysis with AIF1 (Proteintech, 10904-1-AP, 1:500), TNF (Abcam, ab270264,1:150), CD163 (Abcam, ab189915, 1:500), and TIM3 (Abcam, ab241332, 1:500) antibodies were conducted according to our previous procedures [24 (link), 25 (link)]. The protein expression levels were evaluated independently by two experienced pathologists and the scoring criteria refer to our published article [25 (link)].
Ab189915
Manufactured by Abcam
Sourced in United Kingdom
Ab189915 is a primary antibody product manufactured by Abcam. It is designed for use in various laboratory techniques.
Automatically generated - may contain errors
Lab products found in correlation
Mab3174, by R&D Systems (2 mentions)
Ab270264, by Abcam (1 mentions)
Ab241332, by Abcam (1 mentions)
Rabbit anti cd163 monoclonal antibody, by Abcam (1 mentions)
Mouse anti flag monoclonal antibody, by Abmart (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Ecl detection kit, by Bio-Rad (1 mentions)
Polyvinylidene fluoride membrane, by Roche (1 mentions)
Ab20181, by Abcam (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Axioplan 2 microscope, by Zeiss (1 mentions)
K4010, by Agilent Technologies (1 mentions)
Quanto dab brown, by Thermo Fisher Scientific (1 mentions)
M0814, by Agilent Technologies (1 mentions)
M7103, by Agilent Technologies (1 mentions)
Harris hematoxylin, by Merck Group (1 mentions)
Ab16667, by Abcam (1 mentions)
Ab5694, by Abcam (1 mentions)
K3461, by Agilent Technologies (1 mentions)
6 protocols using ab189915
1
Immunohistochemical Analysis of Tumor Markers
2
Western Blot Analysis of CD163 and GAPDH
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Cells were lysed with RIPA lysis buffer containing protease inhibitor (Roche, 04693124001) and cell supernatant were collected after centrifuging in 4°C. Then 30 μg protein were denaturated and separated by SDS-PAGE. Protein in gels was transferred to a PVDF membraned (Millipore, K5HA3225R), followed blocking with 5 % skimmed milk, incubated with primary antibody overnight and incubated with secondary antibody for 1 hour at room temperature. Finally, membrane were detected with enhanced chemiluminescence reagent (Thermo Fisher Scientific, PK210376). The following antibodies were used: rabbit anti-CD163 monoclonal antibody (1;1000, Abcam, ab189915); mouse anti-flag monoclonal antibody (1:5000, Abmart, 224084); mouse anti-GAPDH monoclonal antibody (1:5000, Kangcheng, kc-5G4).
3
Western Blot Analysis of Macrophage Markers
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Cell lysates were extracted in RIPA lysis buffer containing protease inhibitor cocktail and protein phosphatase inhibitor cocktail for 20 minutes on ice. Total cell lysates were clarified by centrifugation. Protein concentration was determined using a DC protein assay kit (Bio-Rad, Hercules, CA, USA) following the manufacturer’s protocol. Protein samples (30 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis through 4% to 20% gradient gels and transferred to a polyvinylidene fluoride membrane (Roche, Mannheim, Germany), which was blocked with 5% skim milk in tris-buffered saline with polysorbate 20 for 1 hour at room temperature. Primary antibodies against CD163 (ab189915; Abcam), HLA-DR (ab20181; Abcam), and β-actin (Cell Signaling Technology, Inc.) were diluted 1:1,000 in TBST. The membranes were incubated overnight at 4°C with the primary antibodies and then washed three times with TBST before they were incubated in 1:5,000 horseradish peroxidase-conjugated goat anti-rabbit IgG antibody or goat anti-mouse IgG antibody (Bio-Rad) for 1 hour at room temperature. The membrane was washed in TBST, and the hybridized protein bands were detected with an ECL detection kit (Bio-Rad) and processed with Image Lab software (Bio-Rad).
4
Quantifying Immune Cell Infiltrates in Skin
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Immunohistochemical stainings with CD163 for macrophages (1:400 overnight 4° C, monoclonal rabbit anti‐CD163 [C‐terminal], Abcam, ab189915, EPR14643‐36, Cambridge, UK) and myeloperoxidase (MPO) for neutrophils (1:400 overnight 4° C, monoclonal mouse‐anti‐human‐myeloperoxidase antibody, R&D Systems, Inc., MAB3174, 392,105, Minneapolis, USA) as well as a routine toluidine‐blue‐staining for mast cell numbers (0.5% aqueous toluidine blue solution for 24 h) were performed from paraffin‐embedded lesional skin samples (MPO: UV = 36, CSU = 27; CD163: UV = 35, CSU = 27; Toluidine blue: UV = 20, CSU = 18), each section cut at 5 µm. An immunohistochemistry protocol with Polymer‐labelled secondary antibodies (anti‐mouse: EnVision+/HRP mouse, Dako, K400011‐2, Glostrup, Denkmark; anti‐rabbit: EnVision+/HRP rabbit, Dako, K4010, Glostrup, Denmark; both applied for 30 min at room temperature) and AEC + ‐substrate system (Dako, K346111‐2, Glostrup, Denmark, applied for 10 min at room temperature) was used for detecting the primary antibodies. For the immunohistological stainings, photos were taken by an Axioplan‐II‐microscope (Zeiss) at 200× magnification and % of positivity of stained area was assessed by Fiji software.
5
Immunohistochemical Profiling of Tissue Samples
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Slides were de-paraffinized in xylene and rehydrated through graded ethanol followed by heat mediated antigen retrieval using 10 mM Sodium Citrate Buffer (pH 6.0). IHC was performed via incubations with one primary antibody followed by host-matched secondary polymer reagent and color substrate. Primary antibodies used were as follows: CD3+, CD8+, HLA-DR, CD68 (A0452, M7103, M0746, M0814 – Dako), CD163, Ki-67, and αSMA (ab189915, ab16667, ab5694 – Abcam), cleaved-Caspase-3 (#9661, Cell Signal Technologies), and FOXP3 (14–4777–82, eBioscience). Secondary reagents were ImmPress Rabbit HRP and Mouse HRP (Vector Laboratories). Color development was performed using Quanto DAB brown (Fisher Scientific). Slides were counterstained with Harris hematoxylin (Sigma) as appropriate, dehydrated and mounted.
6
Immunohistochemical Analysis of FACAS Skin
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Paraffin sections (5 µm) of a FACAS patient skin sample and control skin were prepared as explained above, and processed for routine (hematoxylin/eosin) and immunohistological stainings. We used an immunohistochemistry protocol (streptavidin–biotin labeling) employing primary antibodies targeting MPO (MAB3174, R&D Systems) 1:400 overnight at 4 °C or CD163 (ab189915, Abcam) 1:400 overnight at 4 °C. After washing 3 × 5 min with TBS, REAL Detection System Alkaline Phosphatase/RED (K5005, Dako) for MPO staining or labeled polymer-HRP anti-rabbit (K4011, Dako) together with AEC + high sensitivity substrate chromogen ready-to-use (K3461, Dako) for CD163 staining were applied according to the manufacturer’s instructions, and subsequently analyzed by bright-field microscopy. Samples of tonsil tissue served as positive controls. Sections omitting the primary antibody served as negative controls.
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