Sections of 5 mm-thickness were obtained from tissues embedded in paraffin by using a microtome. After dewaxing in xylene, washing in ethanol series and rehydrating in water, sections were processed as shown below. All samples were processed simultaneously.
1. For histological analysis of tissue structure, tissue sections were stained with Masson's trichrome staining method. Briefly, samples were incubated in solution A –0.5 ml acid fuchsin, 0.5 ml glacial acetic acid and 99 ml distilled water- for 15minutes, in solution B -1 g phosphomolybdic acid and 100 ml distilled water- for 10 minutes and in solution C – 2 g methyl blue dye, 2.5 ml glacial acetic acid and distilled water up to 100 ml- for 5 minutes. Then, samples were washed in distilled water, dehydrated in alcohol and xylene and mounted for light microscopy analysis.
2. To determine the number of cells per area of tissue (cell density analysis), tissue sections were stained with 4,6-diamidino-2-phenylindole (DAPI) and analyzed using a light microscope. All cell nuclei were automatically quantified using the Image J software.
3. To analyze the fibrillar components of the ECM by histochemistry, samples were stained as follows [14] (link):
– To evaluate the presence of collagen fibers, tissues were stained with the Picrosirius method using Sirius red F3B reagent for 30 min and counterstained with Harris' Hematoxylin for 5 min. To analyze the three-dimensional collagen fiber organization, samples stained with Picrosirius were evaluated using a polarized Nikon Eclipse 90i light microscope.
– For reticular fibers, tissues were stained with the Gomori's reticulin metal reduction method using 1% potassium permanganate for 1 min, followed by 2% sodium metabisulphite solution and sensibilization with 2% iron alum for 2 min. After that, samples were incubated in ammoniacal silver for 10–15 min and in 20% formaldehyde for 3 min. Finally, differentiation was performed with 2% gold chloride for 5 min and 2% thiosulphate for 1 min. No counterstaining agent was used.
– To evaluate elastic fibers, the orcein method was used. All samples were incubated in the orcein solution for 30 min at 37°and differentiated in acid-alcohol for a few seconds. No counterstaining agent was used.
4. To analyze the non-fibrillar components of the ECM, samples were stained as follows [14] (link):
– To determine the glycoproteins content in each tissue type, we used the Schiff Periodic acid staining method (PAS). Briefly, 0.5% periodic acid solution was used for 5 min as oxidant, followed by incubation in Schiff reagent for 15 min. Samples were slightly counterstained with Harris's hematoxylin for 20 seg.
– For analysis of proteoglycans, each tissue section was incubated in alcian blue solution for 30 min and then counterstained with nuclear fast red solution for 1 min.
1. For histological analysis of tissue structure, tissue sections were stained with Masson's trichrome staining method. Briefly, samples were incubated in solution A –0.5 ml acid fuchsin, 0.5 ml glacial acetic acid and 99 ml distilled water- for 15minutes, in solution B -1 g phosphomolybdic acid and 100 ml distilled water- for 10 minutes and in solution C – 2 g methyl blue dye, 2.5 ml glacial acetic acid and distilled water up to 100 ml- for 5 minutes. Then, samples were washed in distilled water, dehydrated in alcohol and xylene and mounted for light microscopy analysis.
2. To determine the number of cells per area of tissue (cell density analysis), tissue sections were stained with 4,6-diamidino-2-phenylindole (DAPI) and analyzed using a light microscope. All cell nuclei were automatically quantified using the Image J software.
3. To analyze the fibrillar components of the ECM by histochemistry, samples were stained as follows [14] (link):
– To evaluate the presence of collagen fibers, tissues were stained with the Picrosirius method using Sirius red F3B reagent for 30 min and counterstained with Harris' Hematoxylin for 5 min. To analyze the three-dimensional collagen fiber organization, samples stained with Picrosirius were evaluated using a polarized Nikon Eclipse 90i light microscope.
– For reticular fibers, tissues were stained with the Gomori's reticulin metal reduction method using 1% potassium permanganate for 1 min, followed by 2% sodium metabisulphite solution and sensibilization with 2% iron alum for 2 min. After that, samples were incubated in ammoniacal silver for 10–15 min and in 20% formaldehyde for 3 min. Finally, differentiation was performed with 2% gold chloride for 5 min and 2% thiosulphate for 1 min. No counterstaining agent was used.
– To evaluate elastic fibers, the orcein method was used. All samples were incubated in the orcein solution for 30 min at 37°and differentiated in acid-alcohol for a few seconds. No counterstaining agent was used.
4. To analyze the non-fibrillar components of the ECM, samples were stained as follows [14] (link):
– To determine the glycoproteins content in each tissue type, we used the Schiff Periodic acid staining method (PAS). Briefly, 0.5% periodic acid solution was used for 5 min as oxidant, followed by incubation in Schiff reagent for 15 min. Samples were slightly counterstained with Harris's hematoxylin for 20 seg.
– For analysis of proteoglycans, each tissue section was incubated in alcian blue solution for 30 min and then counterstained with nuclear fast red solution for 1 min.
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