For fluorescence immunohistology, formalin-fixed paraffin embedded skin biopsy sections were dewaxed in xylene and rehydrated through descending ethanol concentrations. Antigen retrieval was performed by heating the sections at 100°C in citrate-based buffer, pH 6 (Vector laboratories, H-3300) for 20 min. Samples were then blocked in TBS-T (20 mM Tris, 140 mM NaCl, pH 7.6, 1% Tween 20 [Sigma, T9416]) supplemented with 5% BSA and 0.1 M glycine for 45 min at RT before overnight staining in primary antibodies diluted in blocking buffer at 4°C. Following washes in TBS-T, samples were incubated with Alexa Fluor 488 donkey anti-rabbit IgG (H+L) and/or TRITC donkey anti-sheep IgG (H+L) (Jackson ImmunoResearch, 711–545–152 and 713–025–147, respectively) secondary antibodies (1:400 in blocking buffer) for 45 min at RT. DNA was stained with DAPI (1 µg/mL) before mounting coverslips with ProLong® Gold antifade mounting medium. Confocal imaging of fixed samples was performed as above. All image processing (brightness and contrast) was performed on all pixels in each image.
Alexa fluor 488 donkey anti rabbit igg h l
Manufactured by Jackson ImmunoResearch
Sourced in United Kingdom, United States
Alexa Fluor 488 donkey anti-rabbit IgG (H+L) is a secondary antibody conjugated with Alexa Fluor 488 dye. It is designed to detect and bind to rabbit primary antibodies, recognizing both the heavy (H) and light (L) chains.
Automatically generated - may contain errors
Lab products found in correlation
H 3300, by Vector Laboratories (1 mentions)
Af1623, by R&D Systems (1 mentions)
Vectashield with dapi, by Vector Laboratories (1 mentions)
Af305 na, by R&D Systems (1 mentions)
Af4666, by R&D Systems (1 mentions)
Goat anti endomucin, by R&D Systems (1 mentions)
Aqua poly mount medium, by Polysciences (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
3 protocols using alexa fluor 488 donkey anti rabbit igg h l
1
Fluorescence Immunohistology of Skin Biopsies
2
Immunofluorescence Staining of Murine Placental Tissue
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Placentas at 18.5 dpc were collected and fixed overnight in 4% PFA at 4°C. The tissues were then processed for paraffin embedding. Sections of size 5 μm thickness were sliced,
deparaffinized, and rehydrated. Antigen retrieval was performed by boiling the sections for 20 min in a citrate-based antigen-unmasking solution. Non-specific binding was blocked using 4%
normal horse serum for 30 min.
The following primary antibodies were used: Rabbit anti-Nrk (Merck, HPA017238, 1:100), goat anti-endomucin (R&D Systems, MN, USA, AF4666, 1:200), goat anti-proliferin (R&D Systems,
AF1623, 1:200), and goat anti-IGF1r (R&D Systems, AF305-NA, 1:200), which were incubated overnight at 4°C. The secondary antibodies used were Alexa Fluor® 488 donkey anti-rabbit IgG
H&L (Jackson ImmunoResearch, 711-545-152, 1:500) and Alexa Fluor® 594 donkey anti-goat IgG H&L (Abcam, Cambridge, UK, ab150132, 1:500), which were incubated for 1 h at 20–25°C.
Counterstaining and mounting were performed using VECTASHIELD® with DAPI (Vector Laboratories, H-1200).
deparaffinized, and rehydrated. Antigen retrieval was performed by boiling the sections for 20 min in a citrate-based antigen-unmasking solution. Non-specific binding was blocked using 4%
normal horse serum for 30 min.
The following primary antibodies were used: Rabbit anti-Nrk (Merck, HPA017238, 1:100), goat anti-endomucin (R&D Systems, MN, USA, AF4666, 1:200), goat anti-proliferin (R&D Systems,
AF1623, 1:200), and goat anti-IGF1r (R&D Systems, AF305-NA, 1:200), which were incubated overnight at 4°C. The secondary antibodies used were Alexa Fluor® 488 donkey anti-rabbit IgG
H&L (Jackson ImmunoResearch, 711-545-152, 1:500) and Alexa Fluor® 594 donkey anti-goat IgG H&L (Abcam, Cambridge, UK, ab150132, 1:500), which were incubated for 1 h at 20–25°C.
Counterstaining and mounting were performed using VECTASHIELD® with DAPI (Vector Laboratories, H-1200).
3
Immunofluorescent Staining of Tight Junctions
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Cells in Transwell® inserts were washed twice with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. Cells were washed twice with PBS, then treated with 0.1 M glycine for 15 min and permeabilized with 0.1% Triton X-100 for three times, 2 min each. Cells were blocked with 10% normal donkey serum for 2 h at room temperature then incubated with rabbit polyclonal anti- zonula occludens-1 (ZO-1) (1:100, #61-7300, Thermo Fisher Scientific, Waltham, MA, United States) overnight at 4°C. Cells were washed three times in PBS and incubated with Alexa Fluor® 488 donkey anti-rabbit IgG (H + L) for 1 h (1:500, #711-546-152, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, United States). Nuclei were counterstained with Hoechst 33342 (1 μg/ml, #B2261, MilliporeSigma, Burlington, MA, United States). The Transwell® membranes with cells were mounted on microscope slides with Aqua-Poly/Mount medium (#18606-20, Polysciences, Warrington, PA, United States). Images were acquired using a Leica SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany).
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