Samples of fetal lungs were taken from three different litters from each group by random sampling (n = 3/group). Paraffin sections were placed in an oven at 65 °C for 2 h, dewaxed, and washed three times with PBS (5 min each time). Antigens were retrieved in EDTA buffer in a low‐power microwave until boiling. After natural cooling, sections were washed thrice with PBS (5 min each time). Peroxidase was eliminated with 3% hydrogen peroxide solution at room temperature for 10 min. Sections were washed in PBS thrice, 5 min each time, and treated with 5% BSA for 20 min. Sections were stained with 100 μL (1 : 200) of diluted primary antibody against SP‐B (ABS21; Millipore corp., Billerica, MA, USA) or SP‐C (sc‐13979; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C overnight. After washing with PBS thrice (5 min each time), 100 μL (1 : 200) of secondary antibody (HRP‐labeled goat anti‐rabbit, AS‐1107) was added and incubated at 37 °C for 50 min. Antibodies were revealed with 100 μL of fresh DAB solution. The sections were observed by light microscopy (Olympus, Tokyo, Japan).
Sc 13979
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-13979 is a laboratory instrument designed for molecular biology applications. It is capable of performing precise temperature control and thermal cycling, making it suitable for techniques such as PCR (Polymerase Chain Reaction). The product specifications and technical details are available upon request.
Automatically generated - may contain errors
Lab products found in correlation
Light microscopy, by Olympus (1 mentions)
Ventana discovery, by Roche (1 mentions)
M0876, by Agilent Technologies (1 mentions)
Bond 3 staining system, by Leica (1 mentions)
Lsm 510, by Zeiss (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Ab53226, by Abcam (1 mentions)
Ab51891, by Abcam (1 mentions)
Mouse anti vimentin cy3, by Merck Group (1 mentions)
Dmi4000d microscope, by Leica (1 mentions)
Bcip nbt solution, by Beyotime (1 mentions)
Confocal microscope, by Nikon (1 mentions)
C1si inverted microscope, by Nikon (1 mentions)
Formaldehyde, by Solarbio (1 mentions)
Hoechst 33258, by Beyotime (1 mentions)
5 protocols using sc 13979
1
Immunohistochemical Analysis of Fetal Lung Surfactants
2
Immunostaining of Lung Tissues in Rat
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The lungs of the rats were immunostained by the labelled streptavidin-biotin method using the Ventana Discovery staining system (Ventana Medical Systems, Tucson, AZ, USA) for the staining of SP-C, and the Leica BOND-III staining system (Leica Biosystems, Nussloch, Germany) for the staining of napsin A and CD68. The anti-rat SP-C polyclonal antibody (1:50 dilution; sc-13979, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); anti-rat napsin A monoclonal antibody (1:100 dilution; NCL-N-Napsin A, Leica Biosystems Newcastle Ltd., Newcastle Upon Tyne, UK); and anti-rat CD68 monoclonal antibody (1:100 dilution; M 0876, DakoCytomation, Glostrup, Denmark) were used. In our previous study35 (link)–37 (link), the expression of napsin A in the alveolar walls was used to identify inflammatory changes, such as hyperplasia and lesions (negative for napsin A), which were thought to progress to neoplastic lesions (positive for napsin A; hyperplasias, adenomas, and adenocarcinomas). SP-C localises in type II alveolar cells48 (link). In our previous study, we reported that high expression of SP-C is a potential marker of proliferative lesions including hyperplasia35 (link). CD68 was used as a marker for macrophages because it is highly expressed by monocytes.
3
Immunofluorescence analysis of lung cells
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Primary AEC isolated from lung biopsy were fixed in 3% paraformaldehyde (PFA) for 10 minutes at room temperature, washed three times in 0.1% glycin/PBS, and incubated with either rabbit polyclonal antibody to E-cadherin (1:200) (#ab53226) (Abcam, USA), or rabbit polyclonal SP-C (1:50) (#sc13979) (Santa Cruz Biotechnology, USA) or mouse monoclonal antibody to surfactant protein A (SP-A) (1:200) (#ab51891) (Abcam, USA) over night in a moist chamber at 4 °C. For detection Cy3 labelled anti rabbit secondary antibody was used for E-cadherin and SP-C, while a alkaline phosphatase labeling was used to detect SP-A. A549 cells were treated with TN or TG, for 18 hours and 7 days respectively and grown in either BMSC-cm or normal media for 24 hours. Cells were then fixed in 3% PFA as described above and subsequently stained overnight at 4 °C in a moist box with anti-cytokeratin, pan-FITC anti-mouse (1:200) (#C5992), mouse anti-vimentin-Cy3 (1:1000) (both: Sigma, USA) (#C9080) or caspase-3 (1:200) (#9662 S) (Cell Signaling, USA). Samples were then washed 3 times with PBS and analyzed under a confocal microscope LSM 510 Carl Zeiss or under Leica DMI4000D microscope.
4
TUNEL Assay for Apoptosis Detection
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Thin paraffin-embedded lung sections were used for the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay commercial kit (Cat. No. G3250; Promega) using the manufacturer’s instructions. Positive and negative control sections were prepared with either label solution only or by pre induction of strand breakage using recombinant DNase I. The reactions were terminated, and the slides were stained for surfactant protein C (SP-C; SC-13979; Santa Cruz Biotechnology) and treated with primary antibody followed by Alexa fluorescent secondary antibody (Alexa 488). Nuclei were counterstained with DAPI. Sections from four to five lungs in each group were stained, and at least five unique fields per section were quantified in each experiment by a blinded observer using Nikon image software (NIS element BR 5.2).
5
Alkaline Phosphatase and Surfactant Protein C Staining Protocol
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For the alkaline phosphatase (BCIP/NBT) staining, cells were fixed in 4% formaldehyde (Beijing Solarbio Science & Technology, Co., Ltd.) at room temperature for 10 min. Following washing with phosphate-buffered saline (PBS) three times, cells were incubated with 3 ml BCIP/NBT solution (Beyotime Institute of Biotechnology, Shanghai, China) for 10 min at room temperature. Cells were then rinsed with water, blotted dry and imaged using a C1si inverted microscope (Nikon Corporation, Tokyo, Japan).
Immunofluorescence of surfactant protein C (SP-C) was used to verify AECII. SP-C rabbit anti-mouse polyclonal antibody (1:100; sc-13979; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was incubated at 4°C for 15 h, then TRITC-conjugated goat anti-rabbit polyclonal antibody (1:200; 85851; Jackson ImmunoResearch Laboratories, Inc., West Grove, BA, USA) was added and incubated at 37°C for 1 h. Hoechst 33258 (Beyotime Institute of Biotechnology) was added to label the nuclei. A confocal microscope (Nikon Corporation) was used to observe the cells. The present study was approved by the Ethics Committee of Beijing Military General Hospital (Beijing, China) and conformed to the guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health (23 ).
Immunofluorescence of surfactant protein C (SP-C) was used to verify AECII. SP-C rabbit anti-mouse polyclonal antibody (1:100; sc-13979; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was incubated at 4°C for 15 h, then TRITC-conjugated goat anti-rabbit polyclonal antibody (1:200; 85851; Jackson ImmunoResearch Laboratories, Inc., West Grove, BA, USA) was added and incubated at 37°C for 1 h. Hoechst 33258 (Beyotime Institute of Biotechnology) was added to label the nuclei. A confocal microscope (Nikon Corporation) was used to observe the cells. The present study was approved by the Ethics Committee of Beijing Military General Hospital (Beijing, China) and conformed to the guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health (23 ).
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