The lung tissue sections were deparafnized in a xylene series and rehydrated through a decreasing ethanol series for immunofluorescence staining. The slides were pretreated by microwave in citrate buffer (100 mM, pH 7.0) for 10 min and washed three times with PBS. H2O2 at 3% was used to eliminate the endogenous peroxidase activity. The slides were washed with PBS and incubated with blocking solution for 30 min at room temperature. They were then incubated overnight at 4°C in anti-collagen I antibody dilution (1:50) from Santa Cruz Biotechnology and anti-fibronectin antibody dilution (1:100) from Proteintech Group. Slides were washed with PBS and incubated with the secondary antibody goat anti-rabbit Cy3 (1:200) from Proteintech Group for 1 h at room temperature in the dark. The nucleus was stained with DAPI. The samples were then washed three times with PBS and then analyzed under a fluorescence microscope (Olympus DP72, Tokyo, Japan).
Goat anti rabbit cy3
Manufactured by Proteintech
Goat anti-rabbit Cy3 is a secondary antibody conjugated with the fluorescent dye Cy3. It is designed to detect and visualize rabbit primary antibodies in various immunoassays and imaging techniques.
Automatically generated - may contain errors
Lab products found in correlation
Anti fibronectin antibody, by Proteintech (1 mentions)
Dm4000 fluorescence microscope, by Leica camera (1 mentions)
Goat anti mouse fitc, by Proteintech (1 mentions)
Dapi containing mounting medium, by Vector Laboratories (1 mentions)
Calponin, by Abcam (1 mentions)
Elastin, by Bioss Antibodies (1 mentions)
Collagen 3, by Bioss Antibodies (1 mentions)
Lamc1, by Cell Signaling Technology (1 mentions)
Mmp 9, by Abcam (1 mentions)
Collagen 1, by Abcam (1 mentions)
Vimentin, by Abcam (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Gapdh, by Proteintech (1 mentions)
4 protocols using goat anti rabbit cy3
1
Immunofluorescence Analysis of Lung Tissue
2
Immunofluorescence Analysis of NLRP3 and UCHL5
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Cells were fixed in 4% paraformaldehyde before being blocked in PBS with 10% normal goat serum and 0.1% Triton X-100 for 30 minutes, and then stained overnight with primary antibody against NLRP3 (R&D, Cat#MAB7578) and UCHL5 (Abcam, Cat#ab133508) at 4 °C. After washing with PBS for 30 minutes, cells were incubated with goat anti-rat FITC (Proteintech, Cat#SA00003-11) and goat anti-rabbit Cy3 (Proteintech, Cat#SA00009-2) at room temperature. Cells were stained with DAPI and images were captured with a Leica DM4000 fluorescence microscope.
3
Immunohistochemical and Immunofluorescence Analysis
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The deparaffinized sections were subjected to heat mediated antigen retrieval, and blocked in H2O2 for 30 minutes, followed by PBS with 5% BSA and 0.2% Triton X-100 at room temperature for 30 minutes, and then stained overnight with primary antibody against NLRP3 (Abcam, Cat#ab214185), Prx1 (Origene, Cat#TA803116), USP1 (CST, Cat#D37B4) or UCHL5 (Abcam, Cat#ab133508) at 4 °C. For immunohistochemistry staining, after rinsing with PBS for 15 minutes, tissues were incubated with HRP-conjugated secondary antibody at room temperature. Sections were then washed and colors were developed with DAB (3,3ʹ-diaminobenzidine). Next, hematoxylin was used as a counterstain. For immunofluorescence staining, tissues were incubated with goat anti-mouse FITC (Proteintech, Cat#SA00003-1) or goat anti-rabbit Cy3 (Proteintech, Cat#SA00009-2) at room temperature. Slides were mounted with Mounting Medium containing DAPI (Vector Labs, Burlingame, CA, USA). All the images were captured with a Leica DM4000 fluorescence microscope.
4
Protein Expression Analysis of Stem Cells
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A radio-immunoprecipitation assay (RIPA) lysis buffer (Yisheng Biological Technology Co., Ltd., Shanghai, China) including proteinase suppressors was applied to isolate total protein. The concentration of total protein was detected by employing bicinchoninic acid (BCA) method (ComWin Biotech Co., Ltd., Beijing, China). A 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate 20 µg of protein samples. Next, the samples were put onto a polyvinylidene difluoride (PVDF) membrane. After being blocked with 5% non-fat milk for 1 hour, the membrane was incubated with the antibodies overnight at 4 °C. The results were scanned by Quantity One software (version 4.6.9, Bio-Rad Laboratories, Hercules, CA, USA). The antibodies used in this part were as following, CD90 (Invitrogen Catalog #MA1-80650); CD44 (Invitrogen Catalog #MA5-17520); CD45 (Invitrogen Catalog #12-0461-82); Cav1 (Bioss,bs-1453R); MyHC (proteintech, 22281-1-AP); Goat anti-rabbit cy3 (proteintech SA00009-2); GAPDH (proteintech, 60004-1-Ig); MyoD (proteintech, 18943-1-AP); Collagen III (Bioss, bs-0549R); Collagen I (abcam, ab260043); LAMC1 (cell signaling, #92921); MMP1 (Affinify, DF6325); MMP9 (abcam, ab76003); HOXA11 (Bioss, bs-666R); Elastin (Bioss, bs-1756R); Calponin (abcam, ab46794); Vimentin (abcam, ab92547); GFP (abcam, ab1218).
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