An immunohistochemical staining for Ki-67 was performed using the HRP-polymer technique, using the ImmPRESS Anti-Rabbit kit (Vector Laboratories, Burlingame, CA.) Formalin-fixed, paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated through graded ethanol. After heat-induced epitope retrieval and endogenous peroxidase blocking nonspecific sites were blocked by incubating lung slides for 30 min with rodent block M (Biocare Medical, Concord, CA) and human Mucil-Air HF sections with 2.5% normal horse serum. The lung sections were then incubated with rabbit Ki-67 antibody (CRM325C, Biocare Medical, Concord, CA), and Mucil-Air HF sections were incubated with rabbit polyclonal anti-Ki-67 (SP6) antibody at a 1:100 dilution for 30 min at room temperature. Normal rabbit IgG (Calbiochem, Billerica, MA) at the equivalent dilution was applied to the negative control in lieu of antibody. The antigen–antibody complex was detected using either rabbit on rodent HRP polymer (Biocare Medical, Concord, CA) and 3,3-diaminobenzidine (DAB) chromogen (Dako North America, Inc., Carpenteria, CA). Slides were then counterstained with hematoxylin.
Immpress anti rabbit kit
Manufactured by Vector Laboratories
Sourced in United States
The ImmPRESS Anti-Rabbit kit is a secondary detection system used in immunohistochemistry and immunocytochemistry applications. It contains a pre-diluted, ready-to-use peroxidase-conjugated anti-rabbit IgG secondary antibody, which can be used to detect and visualize rabbit primary antibodies in tissue or cell samples.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Rodent block m, by Biocare Medical (1 mentions)
Rodent hrp polymer, by Biocare Medical (1 mentions)
Crm325c, by Biocare Medical (1 mentions)
Anti gfp antibody, by Abcam (1 mentions)
Methyl green, by Vector Laboratories (1 mentions)
Bloxall, by Vector Laboratories (1 mentions)
Mouse igg2b isotype control, by R&D Systems (1 mentions)
Cell lysis buffer, by Merck Group (1 mentions)
Goat anti c3d antibody, by R&D Systems (1 mentions)
Anti goat immpress kit, by Vector Laboratories (1 mentions)
4 protocols using immpress anti rabbit kit
1
Immunohistochemical Ki-67 Staining Protocol
2
GFP Immunohistochemistry in Rat Tissue
3
Immunohistochemical Staining of IL-26 and CD68 in BAL Cells
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The ICC staining was performed using cytospin slides prepared with freshly isolated BAL cells harvested from LTRs. These cytospin samples were air-dried and frozen (− 80 °C) until further processing. For study processing, the cytospin slides were thawed and fixed in formaldehyde (4%). To reduce unspecific staining, the slides were incubated first with protein serum-free block (Dako® Agilent Technologies, Denmark), followed by horse serum (5%), and, subsequently, by BLOXALL (Vector Laboratories® Ca, USA). After this, the slides were incubated with primary monoclonal mouse anti-human IL-26 antibodies (5 ug/ml) (Clone 197505, R&D Systems Inc., Mi, USA) or mouse IgG2b isotype control (Clone 20116, R&D Systems Inc.). After washing, slides were incubated with a secondary antibody from the anti-mouse immPRESS kit (5ug/l) (Vector Laboratories®). Bound antibodies were then visualized by ImmPACT VIP-substrate chromogen system (Vector Laboratories®). The procedure was then repeated with in-between washes, but with a primary rabbit anti-human CD68 antibody (5ug/ml) (Abbiotech® Ca, USA), followed by a secondary antibody from the anti-rabbit immPRESS kit (Vector Laboratories®). Bound antibodies were now visualized by ImmPACT DAB-substrate chromogen system (Vector Laboratories®), and slides were finally counterstained with Methyl Green (Vector Laboratories®).
4
Quantifying Complement Activation Markers
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Complement activation was determined by analysis of the complement activation products C3d, C5a, and MAC. To determine C3d deposition, immunohistochemistry staining was performed on paraffin-embedded tissue sections as previously described (Moseley et al., 2010 (link)). In brief, proteinase K enzyme antigen retrieval (Dako) was performed for 5 min at room temperature followed by peroxidase and serum blocking steps. Goat anti-C3d antibody (R&D Systems) was diluted 1:40 in PBS and incubated on slides for 2 h at room temperature, followed by antibody detection with anti–goat ImmPRESS kit (Vector Laboratories). MAC deposition was visualized by IHC on paraffin-embedded tissue using rabbit anti–rat C5b-9 antibody (1:1,000, courtesy of P. Morgan), followed by anti–rabbit ImmPRESS kit (Vector Laboratories). For C5a determinations, liver homogenates were prepared from frozen liver samples homogenized in cell lysis buffer (Sigma-Aldrich) containing a protease inhibitor cocktail (Thermo Fisher Scientific). Homogenates were centrifuged at 10,000 g for 10 min at 4°C, and C5a levels in supernatant determined using an ELISA kit according to the manufacturer’s instructions (R&D Systems, BD). Infiltrating neutrophils were quantified by MPO content of liver samples using a MPO ELISA kit (Hycult Biotechnology) according to the manufacturer’s instructions.
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