We mainly used paraffin-embedded lung sections for immunofluorescent staining. The paraffin sections were dewaxed with xylene, ethanol, and double distilled water, antigen-retrieved with citrate buffer using microwave oven, and blocked with 10% normal goat serum for 2 hours. Then, they were incubated with primary antibodies against alpha smooth muscle actin (α-SMA), Ki-67, Bcl-2, Bax, Caspase-3, P53, mTOR, and Beclin-1, respectively, overnight at 4°C. On the second day, the sections were incubated with relevant fluorescent secondary antibodies (Keygen Biotech Co., Ltd., Jiangsu, China) at 37°C for 2 h and mounted with antifade mounting medium containing DAPI (Solarbio Science & Technology Co., Ltd., Beijing, China). At least 3 views with airway were analyzed per rat in fluorescence microscope. All measurements were performed with the Nikon ECLIPSE 80i biomicroscope and NIS-Elements BR 3.2 image analysis system (Nikon, Japanese). The description and concentration of primary antibodies used in IF analysis are listed in Table 1 .
Antifade mounting medium containing dapi
Manufactured by Solarbio
Sourced in China
Antifade mounting medium containing DAPI is a solution designed to preserve fluorescent signals in biological samples during microscopy. It includes the nuclear stain DAPI, which binds to DNA and emits blue fluorescence upon excitation. This product helps maintain the integrity and visibility of fluorescently labeled samples.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse 80i biomicroscope, by Nikon (1 mentions)
Nis elements br 3.2 image analysis system, by Nikon (1 mentions)
Fluorescent secondary antibodies, by Keygen Biotech (1 mentions)
Anti gfap, by GeneTex (1 mentions)
Anti iba 1, by Affinity Biosciences (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Anti neun, by Abcam (1 mentions)
Mouse anti cd86, by Santa Cruz Biotechnology (1 mentions)
Goat anti cd206, by R&D Systems (1 mentions)
Triton x 100, by Solarbio (1 mentions)
Rabbit anti p65, by Abcam (1 mentions)
3 protocols using antifade mounting medium containing dapi
1
Immunofluorescent Staining of Lung Tissue
2
Immunohistochemical Analysis of Spinal Cord Injury
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After the pigs were sacrificed, spinal cord sections (1 cm) from the central DSCI lesion region were collected and embedded in paraffin. Spinal cord sections (5 μm thick) from each specimen were deparaffinized with xylene and incubated in graded concentrations of ethanol. They were then washed with phosphate-buffered saline (PBS) for 3 × 5 min. The sections were incubated for blocking with a blocking solution (0.1% Triton X-100 in PBS and 10% normal goat serum) at room temperature for 2 h. The sections were incubated overnight with primary antibodies at 4°C. The primary antibodies used were anti-GFAP (1: 2000, GeneTex), anti-Iba-1 (1:200, Affinity), and anti-NeuN (1:100, Abcam). After rinsing with PBS for 3 × 5 min, the sections were incubated with secondary antibodies for 2 h at room temperature. The secondary antibody used in this study was goat anti-rabbit antibody (Alexa Fluor®594) (1:200, Abcam). Following three rinses with PBS, a drop of antifade mounting medium containing DAPI (Solarbio Biotechnology, China) was placed on each slide. Finally, a coverslip was placed on top of each sample. Immunofluorescence imaging was carried out using an Olympus fluorescence microscope.
3
Evaluating NF-κB p65 Expression in RAW264.7 Cells
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Immunofluorescence staining of RAW264.7 cells was carried out to evaluate the level of protein expression and the distribution of NF-κB p65. We seeded 1 × 10 3 cells per well in 24-well plates containing glass coverslips. The test cells were subjected to treatment with GLPS for 24 h. After the treatment and removal of medium, we fixed the cells with 4% paraformaldehyde for 15 min and then permeabilized them in 0.1% Triton X-100 (Solarbio, China) for 10 min. Subsequently, nonspecific binding sites were blocked at RT for an hour in 5% bovine serum albumin (BSA) buffer (Solarbio, China). After rinsing three times with PBST (PBS containing 0.1% Tween-20), the cells were incubated with mouse anti-CD86 (diluted 1 : 200, Santa Cruz, USA), goat anti-CD206 (diluted 1 : 200, RD, USA), and rabbit anti-P65 (diluted 1 : 200, Abcam, UK) overnight at 4 degrees Celsius. Then, we stained the cells with the corresponding secondary FITC-conjugated antibodies (diluted 1 : 200, Proteintech, USA) or PE-conjugated antibodies (diluted 1 : 200, Proteintech, USA) at RT for an hour in the dark.
Finally, an antifade mounting medium containing DAPI (Solarbio, China) to visualize the nucleus was dripped on the glass slides. The slides were stained for 5 minutes at RT. A fluorescence microscope (IX81, Olympus, Japan) was used to observe and photograph the labeled cells.
Finally, an antifade mounting medium containing DAPI (Solarbio, China) to visualize the nucleus was dripped on the glass slides. The slides were stained for 5 minutes at RT. A fluorescence microscope (IX81, Olympus, Japan) was used to observe and photograph the labeled cells.
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