Whole mount staining of lenses for visualization of lens epithelial cell proteins.
Capsule Digestion
Eye
Immunofluorescence
Lens
Whole-mount
Journal
MethodsX
ISSN: 2215-0161
Titre abrégé: MethodsX
Pays: Netherlands
ID NLM: 101639829
Informations de publication
Date de publication:
2021
2021
Historique:
received:
09
03
2021
accepted:
26
04
2021
entrez:
25
8
2021
pubmed:
26
8
2021
medline:
26
8
2021
Statut:
epublish
Résumé
Whole mount imaging of the lens allows for high spatial resolution visualization of lens epithelial structures by using small molecule fluorescent probes. However, the visualization of specific proteins in lens epithelial cells within whole lenses remains a challenge as the capsule that surrounds the lens does not allow penetration of antibodies. Here we describe a whole mount imaging method that allows us to overcome this challenge by digesting the lens capsules of paraformaldehyde fixed lenses using collagenase. This method enables the penetration of antibodies for effective visualization of proteins in the epithelium of whole lenses.•A limitation to lens whole mount imaging is the ability to visualize specific proteins as the collagen capsule surrounding the lens impedes the penetration of antibodies•This protocol helps overcome this limitation by a light collagenase digestion of the capsule of fixed lenses prior to immunostaining•This method allows for the imaging of specific proteins in the epithelium of the whole lens tissue.
Identifiants
pubmed: 34430272
doi: 10.1016/j.mex.2021.101376
pii: S2215-0161(21)00169-2
pmc: PMC8374519
doi:
Types de publication
Journal Article
Langues
eng
Pagination
101376Subventions
Organisme : NEI NIH HHS
ID : R01 EY017724
Pays : United States
Informations de copyright
© 2021 The Author(s).
Références
Exp Eye Res. 2018 Oct;175:148-158
pubmed: 29932883
Prog Retin Eye Res. 2010 Mar;29(2):135-43
pubmed: 20006728
J Cell Sci. 2018 Nov 29;131(23):
pubmed: 30333143
J Clin Invest. 1998 Feb 1;101(3):625-34
pubmed: 9449696
Exp Eye Res. 2009 Feb;88(2):151-64
pubmed: 18773892
Invest Ophthalmol Vis Sci. 2018 Oct 1;59(12):4986-4997
pubmed: 30326070
Matrix Biol. 2010 Apr;29(3):228-36
pubmed: 20026402
Invest Ophthalmol Vis Sci. 2003 May;44(5):2094-102
pubmed: 12714648
Matrix Biol. 2020 Aug;90:79-108
pubmed: 32173580
Mol Biol Cell. 2018 Aug 8;29(16):1963-1974
pubmed: 30088796
Am J Pathol. 2006 Jan;168(1):69-79
pubmed: 16400010
Exp Eye Res. 2008 Jul;87(1):64-70
pubmed: 18508048
J Microsc. 1990 Feb;157(Pt 2):239-45
pubmed: 2179561
Philos Trans R Soc Lond B Biol Sci. 2011 Apr 27;366(1568):1250-64
pubmed: 21402584
Invest Ophthalmol Vis Sci. 2019 Sep 3;60(12):3920-3929
pubmed: 31546253
Cells Tissues Organs. 2005;179(1-2):43-55
pubmed: 15942192
J Cell Mol Med. 2014 Apr;18(4):656-70
pubmed: 24495224
Exp Eye Res. 2016 Jan;142:92-101
pubmed: 26003864
Invest Ophthalmol Vis Sci. 2020 Jun 3;61(6):2
pubmed: 32492110
Biosci Rep. 2001 Aug;21(4):537-63
pubmed: 11900326
Aging (Albany NY). 2019 Dec 16;11(24):12497-12531
pubmed: 31844034
Cell Death Dis. 2013 Oct 24;4:e884
pubmed: 24157878
Mol Biol Cell. 2017 Apr 1;28(7):907-921
pubmed: 28209733
Hum Mol Genet. 2020 Jul 29;29(12):2076-2097
pubmed: 32420594
Dev Dyn. 2018 Mar;247(3):340-345
pubmed: 28480986
Invest Ophthalmol Vis Sci. 1995 Jul;36(8):1709-13
pubmed: 7601651