After dewaxing, rehydration and antigen retrieval, the paraffin-embedded rat lung sections were permeabilized with 0.3% Triton X-100 for 30 minutes, and blocked with 3% BSA for another 30 minutes. The primary antibodies (proSP-C and α-SMA) used for immunofluorescence (IF) were obtained from Abcam (Cambridge, MA, USA). Alexa Fluor 488 and Alexa Fluor 594 (Invitrogen) were used as secondary antibodies. The nuclei were stained for three minutes with 4′,6-diamidino-2-phenylindole (DAPI), which was procured from Invitrogen. For each sample, five random fields were randomly captured. The fluorescence density of α-SMA was quantified using the LAS3000 apparatus (Fujifilm, Raytest, Courbevoie, France).
Pro spc
Manufactured by Abcam
Sourced in United States, United Kingdom
Pro-SPC is a laboratory equipment product designed for sample preparation and concentration. It functions as a protein and nucleic acid concentrator, enabling efficient sample concentration for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Mpeg2000 deg, by NOF corporation (3 mentions)
α sma, by Abcam (2 mentions)
Abca3, by Abcam (2 mentions)
Las3000 apparatus, by Fujifilm (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Foxj1, by Thermo Fisher Scientific (1 mentions)
Mab1179, by R&D Systems (1 mentions)
Spe confocal microscope, by Leica (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Endomucin, by Santa Cruz Biotechnology (1 mentions)
Chicken polyclonal anti gfp antibody, by Abcam (1 mentions)
Ttf 1, by Santa Cruz Biotechnology (1 mentions)
E cadherin, by Santa Cruz Biotechnology (1 mentions)
Anti α sma, by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by Boster Bio (1 mentions)
Ecl reagent, by Merck Group (1 mentions)
Goat anti rabbit igg antibody, by Boster Bio (1 mentions)
Goat anti mouse igg antibody, by Merck Group (1 mentions)
8 protocols using pro spc
1
Immunofluorescence Analysis of Rat Lung
2
Immunofluorescence Staining of Tumorspheres
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
KP tumorspheres were either stained in Matrigel or were paraffin-embedded, sectioned and stained as previously described (Huber et al., 2015 (link)). For Matrigel preparations, tumorspheres were fixed in 4% PFA for 40 min, washed 3×5 min, permeabilized with 0.5% Triton X-100 for 30 min then blocked in 4% BSA for 30–60 min. Staining was performed in blocking buffer with 0.05% Triton X-100 and washes were performed with 0.1% Triton X-100. Tumorspheres were imaged using a Leica SPE confocal microscope. The following antibodies were used for IF: BrdU (NeoMarkers, MS-1058-PO, BRD.3 1:200), cleaved caspase-3 (Cell Signaling Technology, 9661 1:300) pro-SPC (Abcam, ab170699: 1:200), CC10 (SantaCruz, 9772, 1:200) FoxJ1 (eBioscience, 14-9965-82, 2A5), RAGE (R&D, 175410, MAB1179, 1:100).
3
Immunohistochemical Analysis of Lung Tissues
4
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue homogenates were centrifuged at 12,000 rpm for 30 minutes at 4°C. Thirty micrograms of protein was separated by 10% SDS-PAGE and electroblotted onto polyvinylidene difluoride (PVDF) membranes, which were blocked with 5% TBST-milk at room temperature for 1 hour, incubated at 4°C overnight with primary antibody (α-SMA, rabbit polyclonal antibody, Abcam, UK; pro-SPC, rabbit polyclonal antibody, Abcam, UK; AKT, rabbit polyclonal antibody, CST, USA; p-AKT, rabbit polyclonal antibody, CST, USA; and β-actin, mouse monoclonal antibody, CMCTAG, USA) in 1% TBST-milk, and incubated for 1 hour with HRP-conjugated secondary antibody (1 : 5000 dilution, goat anti-rabbit IgG antibody, Boster, China; 1 : 3000 dilution, goat anti-mouse IgG antibody, Millipore, USA). Protein bands were subsequently detected using enhanced chemiluminescent (ECL) reagents (Millipore, USA).
5
Immunohistochemical analysis of lung tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The lung prepared as described above was instilled with 1 ml 4% paraformaldehyde in PBS(+) and fixed with 4% paraformaldehyde for 2 days at room temperature. The lung was embedded in 5% Seaplaque agarose (Lonza) or paraffin and then sectioned to 100 μm or 6 μm thickness, respectively, with a microslicer (DOSAKA EM). Paraffin sections were deparaffinized with xylene and ethanol and then rehydrated. The obtained sections were permeabilized with 0.5% TritonX-100 in PBS(+) for 1 h, stained with specific antibodies against HA (C102), FITC, ABCA3 (3C9), or Pro-SP-C (Abcam), followed by Alexa488-labeled or Alexa546-labeled secondary antibodies, and then mounted with ProLong Diamond reagent (Thermo Fisher Scientific). All images were analyzed using LSM710 laser scanning confocal microscopy (Zeiss).
6
Phenotypic characterization of hAE and hAMS cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
hAE and hAMS cells were fixed immediately after isolation with 1% formaldehyde in PBS for 10 min. After two washing steps in 1% BSA (Sigma Aldrich, USA) in PBS, cells were either prior permeabilised for 20 min in Permeabilization Buffer (Affymetrix, USA) for intracellular staining or directly incubated with the respective primary antibodies for 45 min. Cells were washed and incubated with polyclonal Alexa Fluor 488-conjugated goat anti-rabbit or Alexa Fluor RPE-conjugated goat anti-mouse IgG secondary antibodies (1:750, Thermo Fisher Scientific, USA) for 30 min. Isotype controls were incubated with rabbit and mouse IgG. After washing, cells were analysed on a flow cytometer (Cytoflex, Beckman Coulter, USA). SP-A, Oct-4 and pan-CK were obtained from Santa Cruz Biotechnology (USA) and were used in a dilution of 1:100, all the other antibodies (SP-B, pro-SP-C, ABCA3, SSEA-4) were purchased from Abcam (UK) and were diluted 1:250. Every incubation step was performed on ice.
7
Murine Recombinant IL-4 Signaling Pathway
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Murine recombinant IL-4 was obtained from PeproTech (London, UK). Antibodies against collagen type I was purchased from EMD Millipore (Schwalbach, Germany). Antibodies against fibronectin, α-SMA, arginase-1, CD68, MBD2, and pro-SPC were purchased from Abcam (Cambridge, MA, USA), and antibodies against p-Smad2, p-Smad3, STAT6, P85, p-P85, and p-Akt (Thr308) were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies against MBD2, glyceraldehyde-3-phosphate dehydrogenase, p-Akt (Ser473), and p-STAT6 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-mouse F4/80-PerCP/Cy5.5 and CD206–fluorescein isothiocyanate (FITC) were from BioLegend (San Diego, CA, USA), and CD11c–allophycocyanin (APC) were purchased from BD Biosciences (San Jose, CA, USA). Lipidoid (C12-200) was purchased from Xinjiahecheng Medical Chemistry Corporation (Wuhan, Hubei, China). mPEG2000-DEG was purchased from NOF Corporation (Tokyo, Japan). All other reagents were purchased from Sigma-Aldrich (St Louis, MO, USA), unless otherwise stated.
8
Immunohistochemistry of Lung Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lung tissue sections from the patient obtained by open lung biopsy at 5 mo of age were prepared from formalin-fixed paraffin-embedded samples. Control lung tissue was resected for malignancy. Immunohistochemistry was performed with antibodies against proSP-C, SP-B, ABCA3 (Abcam, Cambridge, UK), or proSP-B (Novocastra, Newcastle, UK), according to standard protocols (30) .
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!