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A dot‐blot based assay to quantify collagen from large arteries
Author(s) -
Condezo-Hoyos Luis A.,
España-Caparros Gabriel,
Pablo Angel L López,
Rodríguez-Rodríguez Pilar,
Gutierrez-Arzapalo Perla Y.,
Ancer Jesús,
Gonzalez M. Carmen,
Arribas Silvia M.
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.lb201
Subject(s) - sirius red , elastin , gelatin , chromatography , dot blot , chemistry , staining , membrane , western blot , biomedical engineering , biochemistry , pathology , medicine , dna , gene
AIM To develop a densitometric method based on dot blot and sirius red staining to quantify vascular collagen, using gelatin as standard, compatible with elastin purification. METHODS Aortae and carotid arteries from adult rats were dried 5 min, weighted and incubated in 0.1N boiling NaOH for 45min. NaOH extracts were tested by dot blot on nitrocellulose or PVDF membranes. We established the optimal conditions for: 1) Sirius red concentration, incubation time and temperature, 2) washing time and composition of washing solution, 3) linearity and specificity of the assay. The compatibility of this method with elastin purification and integrity was assessed by confocal microscopy (488 excitation, 520–600 emission nm, autofluorescent properties). RESULTS Optimal conditions for the densitometric method were: 1) Sirius red concentration 0.00125% w/v, 30 min at 4ºC, 2) tricloroacetic acid and acetone included in the washing solution increased signal/noise ratio, being compatible only with PVDF membranes and 3) gelatin concentration linearity range was 0–0.5 g. Collagen content was quantifiable in carotid and aortae NaOH extracts. Purified elastin has a similar organization to intact arteries. CONCLUSION We have developed a quick, simple, cheap and specific method to quantify collagen in vascular samples compatible with elastin purification. FINANCIAL SUPPORT: MICINN (FEM2009‐13434‐C02‐02).