Alveolar spheres were first embedded in Histogel (Thermofisher), then paraffin-embedded and sectioned the same way as PFA-fixed tissue. The sections were stained with Hematoxylin/Eosin for histology, or underwent antigen retrieval and permeabilization with 0.1% Triton-X100 for immune-fluorescent staining. The protocol included blocking by 5% donkey serum (Sigma), incubation with primary antibodies (1:200, Nkx2.1: ab76013, Abcam; E-Cadherin: 610,181, BD Biosciences; Pro-SPC: WRAB-9337, Seven Hills Bioreagents; Surfactant protein B: sc-133,143, Santa Cruz; Aquaporin 5: ab92320, Abcam; Ki67: AB9260, Millipore; AE2, AF933, R&D; GFP, ab6673, Abcam) overnight at 4 °C, washing with PBS for 3 times, incubation with secondary antibodies (1:1000; Alexa Fluor 488 or 546, Life Technologies) for 1 h at room temperature, and mounting with DAPI Fluoromount-G (Fisher Scientific). TUNEL staining was performed following manufacturer's protocol (Promega). Images were captured using Nikon Ti-PFS inverted microscope and Leica SP8 confocal microscope. All the data for quantification of histological and immunofluorescent staining were measured by Fiji.
Aquaporin 5
Manufactured by Abcam
Sourced in United States
Aquaporin 5 is a water-channel protein that facilitates the movement of water across cell membranes. It is primarily expressed in secretory epithelial cells, such as those found in the salivary and lacrimal glands, as well as the airways. Aquaporin 5 plays a crucial role in the regulation of water homeostasis and fluid secretion in these tissues.
Automatically generated - may contain errors
Lab products found in correlation
Donkey serum, by Merck Group (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Ab6673, by Abcam (1 mentions)
Histogel, by Thermo Fisher Scientific (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
E cadherin, by BD (1 mentions)
4 hydroxy 2 nonenal 4 hne, by Abcam (1 mentions)
Grp78, by Santa Cruz Biotechnology (1 mentions)
Gadd153 chop, by Santa Cruz Biotechnology (1 mentions)
Alpha amylase, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Confocal laser scanning microscope, by Olympus (1 mentions)
Acetylated α tubulin, by Abcam (1 mentions)
Eclipse ti microscope, by Nikon (1 mentions)
Pro surfactant protein c, by Abcam (1 mentions)
Deltavision elite, by Cytiva (1 mentions)
Sftpc, by Merck Group (1 mentions)
Alexa fluor 488, by Abcam (1 mentions)
Alexa fluor 594, by Abcam (1 mentions)
Epcam, by Abcam (1 mentions)
5 protocols using aquaporin 5
1
Alveolar Sphere Histology and Immunostaining
2
Immunohistochemical Analysis of Salivary Gland
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Immunohistochemistry was performed in formalin-fixed, paraffin-embedded submandibular gland tissue as described previously [21 (link)]. Antibodies analyzed with immunohistochemistry were alpha-amylase (1:200, anti-mouse, Santa-Cruz Biotechnology, Dallas, TX, USA), aquaporin-5 (1:150, anti-rabbit, Abcam, Cambridge, UK), 4-Hydroxy-2-nonenal (4-HNE) (1:100, anti-rabbit, Abcam, Cambridge, UK), GRP78 (1:100, anti-mouse, Santa-Cruz Biotechnology, Dallas, TX, USA), and GADD153/CHOP (1:100, anti-rabbit, Santa-Cruz Biotechnology, Dallas, TX, USA). Double-label immunofluorescence was performed using anti-goat IP3R2 (1:100, Santa-Cruz Biotechnology, Dallas, TX, USA) and anti-rabbit AQP5 antibody (1:300, Abcam, Cambridge, UK). Sections were incubated with primary antibodies in a humidified chamber overnight at 4 °C. Slides were then rinsed in 1×TBST buffer (DAKO) three times and incubated with fluorescein isothiocyanate (FITC)-conjugated anti-goat secondary antibody (1:300, Sigma) or TRITC-conjugated anti-rabbit secondary antibody (1:300), respectively, for 1 h at room temperature. Slides were then followed by washing with TBST (3 times) and mounted with DAPI (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Fluorescence was visualized using FITC and TRITC channels in a laser scanning confocal microscope (Olympus, Japan).
3
Multiparametric Immunofluorescence Characterization of Lung Cell Phenotypes
4
Immunofluorescence Analysis of Lung Cells
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Lungs were fixed with 4% paraformaldehyde in phosphate-buffered saline. Immunofluorescence staining was performed on cryosections or paraffin sections. Primary antibodies and dilutions were as follows: Sftpc (Millipore, Ab3786, 1:1,000), Aquaporin 5, AQP5 (Abcam, ab78486, 1:1,000), T1a (Sigma, P995, 1:400), Edu (Beyotime, C0081S), Ki67 (Abcam, ab231172, 1:400), Krt5 (Abcam, ab193895, 1:300), EpCAM (Abcam, ab221552, 1:200), CD45 (Biolegend, 103,116, 1:100), Lysotracker (Invitrogen, L7526), and p63 (Abcam, ab735, 1:200). The secondary antibodies were Alexa Fluor 488 (Abcam, ab150081, 1:400) and Alexa Fluor 594 (Abcam, ab150120, 1:400). Images were obtained using a Zeiss confocal microscope and Delta Vision Elite (Applied Precision).
5
Immunohistochemical Staining of Lung Tissues
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Paraffin sections were stained with hematoxylin/eosin (H&E) (Sigma) or immunostained. For immunofluorescence, after deparaffinazion and antigen retrieval process, sections were incubated with primary antibodies according to manufacturer’s instructions after blocking for 1 h at room temperature. The slides were then washed and incubated with the appropriate secondary antibodies and labeling dyes. Secondary antibodies were coupled to Alexa-488, or Alexa-568 fluorochromes. After washing, tissue sections were mounted with Prolong gold with DAPI. Primary antibodies used are the following: SPC (Abcam ab211326), CCSP (Santa Cruz sc-390313), Podoplanin (Abcam ab11936) and Aquaporin 5 (Abcam ab11936).
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