Paraffin sections (4 μm) were dried, deparaffinized, and stained with hematoxylin eosin safranin (HES) (Dako). For immunohistochemical staining, 4 µm paraffin sections were incubated with antigen retrieval citrate-based solution pH = 9 (Vector Laboratories) at 95 °C for 15 min. The sections were incubated in Bloxall blocking solution (Vector Laboratories) for 10 min to inactivate endogenous peroxidase. FC receptor blocking reagent (Innovex Biosciences) was then added for 45 min. All sections were incubated in PBS-10% FCS for 30 min with either anti-lamin A/C antibody (dilution 1/200, clone EPR4100, Abcam) or anti-osterix/SP7 antibody (dilution 1/100, Abcam) diluted in PBS-5% FCS-1.5% BSA overnight incubation at 4 °C. The sections were washed with PBS-0.1% Triton and incubated in ImPRESS Reagent anti-rabbit IgG (Vector Laboratories) for 30 min. Then, the sections were washed with PBS-Triton 0.1% and incubated in HistoGreen substrate solution (Linaris) for 1 min. Counter coloration was performed using Fast Nuclear Red (Vector Laboratories). All sections were analyzed using a Pannoramic Midi II slide scanner and Case Viewer software (3D HISTECH, Ltd.).
Impress reagent anti rabbit igg
Manufactured by Vector Laboratories
Sourced in United States
IMPRESS Reagent anti-Rabbit IgG is a secondary antibody reagent that binds to rabbit immunoglobulin G (IgG) antibodies. It is designed for use in various immunodetection techniques, such as western blotting, ELISA, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Caseviewer software, by 3DHISTECH (1 mentions)
Bloxall blocking solution, by Vector Laboratories (1 mentions)
Anti lamin a c antibody, by Abcam (1 mentions)
Pannoramic midi 2 slide scanner, by 3DHISTECH (1 mentions)
Hematoxylin, by Vector Laboratories (1 mentions)
Vector dab substrate kit, by Vector Laboratories (1 mentions)
Histoclear, by Merck Group (1 mentions)
4 protocols using impress reagent anti rabbit igg
1
Immunohistochemical Analysis of Lamin A/C and Osterix
2
Immunohistochemistry of Placental Tissue
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Immunohistochemistry procedure was performed as previously described5 (link). Briefly, paraffin embedded human placental tissue samples were sectioned, deparaffinized, and hydrated with Histoclear and ethanol (Sigma, St. Louis, MO, USA). Slides then underwent antigen retrieval using 10 mM sodium citrate (pH 6) buffer in a pressure cooker. Sections were blocked using DAKO Dual Endogenous Enzyme Block for Autostainer and 2.5% normal horse serum (Agilent, Santa Clara, CA, USA). Slides were then incubated with primary antibody m28-RNLS (1:500) overnight at 4 °C and secondary antibody IMPRESS Reagent anti-Rabbit IgG (Vector Laboratories, Burlingame, CA, USA). Antibody binding was detected using Vector DAB substrate kit and counterstained with hematoxylin (Vector Laboratories, Burlingame, CA, USA). Images were obtained at 20× and 60× using Echo® Revolve microscope. Staining specificity was determined by labeling with secondary antibody.
3
Placental Immunohistochemistry Protocol
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19 formalin-fixed and paraffin-embedded placental samples from PP (n=5), PNP (n=4), TP (n=5), and TNP (n=5) patients were sectioned and mounted by Yale Histology Core. Slides were deparaffinized and rehydrated in ethanol prior to antigen retrieval using 10 mM citrate-buffered saline in a pressure cooker at 95°C for 20 minutes. Sections were washed with TBS-Tween (1%) with 300 mM NaCl prior to blocking with DAO Dual Endogenous Enzyme Block for Autostainer for 10 minutes followed by blocking with 2.5% horse serum for 1 hour (Agilent, Santa Clara, CA, USA). Slides were incubated with m28-RNLS (1:250 in TBS) overnight at 4°C and incubated in secondary antibody IMPRESS Reagent anti-Rabbit IgG for 45 minutes (Vector Laboratories, Burlingame, CA, USA). Antibody detection was performed with Vector’s ImmPACT DAB substrate kit prior to a hematoxylin counterstain (Vector Laboratories, Burlingame, CA, USA).
4
Immunohistochemistry of Thymus Annexin A5
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Five male rats aged 0, 30, 60, 90, and 180 days were euthanized by cervical dislocation under anesthesia. The body and thymus weights were measured. The thymus was quickly removed and immediately immersed in 4% paraformaldehyde. Thymus samples were shaken overnight at 4°C and rinsed with phosphate-buffered saline overnight. Fixed tissues were dehydrated by immersion in a series of ethanol and xylene solutions and embedded in paraffin. Sections of 4 μm thickness were prepared. Anti-annexin A5 (MS-2, ×5,000) was used as the primary antibody (PRID: AB_2827744; https://antibodyregistry.org/AB_2827744 ). We obtained the antibody from rabbit serum and used it for immunohistochemistry and western blotting [15 (link), 19 (link)]. ImPRESS REAGENT (anti-rabbit IgG) (Vector Laboratories, Funakoshi, Tokyo, Japan) was used for signal detection. The diaminobenzidine substrate solution (Roche, Basel, Switzerland) was added dropwise to the tissue sections. The reaction was carried out at room temperature, and the degree of coloration was observed under a microscope. The color reaction was stopped with distilled water, followed by immersion in hematoxylin solution for approximately 1 min and rinsing with tap water. The specimens were dehydrated and mounted with a sealant (M-X) (Matsunami Glass, Osaka, Japan).
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