Immortalized clones were seeded into chamber slides (5 × 103 (link) cells/chamber) until 30% confluent, washed with PBS, fixed with 75% (v/v) ethanol for 15 minutes, washed, incubated with 4% paraformaldehyde, washed and blocked with 1% (w/v) horse serum albumin for 30 minutes. The cells were incubated with primary rabbit anti-mouse COMP or goat anti-mouse FSP1 or non-immune IgG for 2 hours. After washing, cells were incubated with fluorescent-labeled donkey anti-rabbit-FITC or donkey anti-goat lgG-CFL647 (Santa Cruz, Dallas, TX) secondary antibody (1:2000v/v), respectively for 1 hour. The nuclei were stained with 4-6-Diamidino-2-phenylindole (DAPI). Following further washing, the cells were mounted with cover slips and observed under a fluorescent microscope (Nikon TS100, Tokyo, Japan). A similar protocol was used for staining TMJ disc tissue, with the exception that the sections were serially incubated with both primary antibodies followed by both secondary antibodies.
Donkey anti rabbit fitc
Manufactured by Santa Cruz Biotechnology
Donkey anti-rabbit FITC is a secondary antibody conjugated with the fluorescent dye FITC (Fluorescein isothiocyanate). It is designed to detect and visualize rabbit primary antibodies in various immunological applications such as immunofluorescence, flow cytometry, and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using donkey anti rabbit fitc
1
Immunofluorescence Staining of Immortalized Cells
2
Immunofluorescence Staining of A2aAR in Colon
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IF staining was performed according to the standard protocol using 5-μm-thick transverse-cut paraffin sections from sigmoid colon biopsies. Briefly, paraffin sections were rendered permeable by incubation in 0.1% Triton X-100 for 30 min, blocked with 10% donkey serum albumin at room temperature (RT) for 30 min, followed by incubation with rabbit anti-human A2aAR primary antibody (1:30 dilution, Santa Cruz Biotechnology) overnight at 4 °C. After rinsing three times with phosphate-buffered saline (PBS), the sections were incubated with secondary antibody (Donkey anti-rabbit FITC, 1:100 dilution, Santa Cruz Biotechnology) for 1 h at RT in the dark, and were washed three times with PBS. Negative controls were prepared by omitting the primary antibody. Nuclei were counterstained with DAPI and the coverslips were mounted on slides by using a fluorescent mounting medium. The stained sections were evaluated using a Leica inverted fluorescence microscope.
3
Histological Analysis of Lung Inflammation and IRAK-M Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For histological analysis, formalin-fixed paraffin-embedded lung tissues were sectioned (4 μm) and then stained with haematoxylin and eosin to visualize inflammatory responses and pathological changes in the lung. The stained sections were then imaged and recorded under light systems (AxioVert 40 CFL, AxioCam MRC, and AxioVision LE Image system, Carl Zeiss). The detection of IRAK-M protein was performed using rabbit anti-IRAK-M (ProSci, 2 μg ml−1 for Immunohistochemistry, 10 μg ml−1 for Immunofluorescence) and Donkey anti-rabbit-FITC (Santa Cruz Biotechnology, 5 μg ml−1) in the paraffin section of mouse lung tissue. Blocking IRAK-M peptide (ProSci, cat# 2355 P, 25 μg ml−1) was used for the negative control experiments. Epithelial cells and macrophages were recognized by antibodies of anti-E-cadherin (Santa Cruz Biotechnology, 10 μg ml−1) and anti-F4/80 (Santa Cruz Biotechnology, 10 μg ml−1), respectively and followed by bovine anti-goat TR (Santa Cruz Biotechnology, 8 μg ml−1) incubation. Inflammation score in haematoxylin and eosin staining (Grade; 0 to 3) and IRAK-M protein expression intensity score in immunostaining (Grade; 0 to 4) were validated in a blinded fashion53 (link)54 (link)55 (link)56 (link).
4
Immunofluorescence Analysis of Colon Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IF staining was performed according to standard protocols by using 5-μm thick transverse-cut paraffin sections from colons of control or DSS colitis mice. Briefly, paraffin sections were rendered permeable by incubation in 0.1% Triton X-100 for 30 min; the sections were then blocked with 10% donkey serum albumin at room temperature (RT) for 30 min and incubated with primary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) against A3AR (1:30 dilution), NF-κB p65 (1:50 dilution), nuclear factor-kappaB inhibitor alpha(IκB-α) (1:50 dilution), or phosphorylated-IκB-α (p-IκB-α) (1:50 dilution) overnight at 4°C. After rinsing three times with phosphate buffered saline (PBS), sections were incubated with secondary antibodies (Donkey anti-rabbit FITC, Donkey anti-goat DyLight™405, Donkey anti-mouse Cy™3, 1:100 dilution, Santa Cruz Biotechnology) for 1 h at room temperature in dark, then washed three times with PBS. Negative controls were prepared by omitting the primary antibodies. Finally, coverslips were mounted on slides by using fluorescent mounting medium with DAPI to counter-stain the nuclei. The staining was evaluated on a Leica inverted fluorescence microscope.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!