Application Of Histochemical Staining Methods In The Study Of The Growth Plate Histology

Objective: The growth plate is a highly organized cartilage structure entrapped between the epiphyseal and metaphyseal bone at the distal ends of the long bones. Longitudinal growth takes place by a process called endochondral ossification, in which a cartilaginous scaffold is replaced by bone in a coordinated fashion. The growth plate can be divided into horizontal zones of chondrocytes at different stages of differentiation:the reserve zone,proliferating zone and the hypertrophic zone. In this research, after embedded in the paraffin, we made the normal rats'tibia sample slices. Then the slices were stained by the HE staining, Masson trichrome staining, AB-PAS staining, toluidine blue staining, Safranine staining, AZAN trichrome staining, Van Gieson staining, Mallory trichrome staining, Safranin'O staining, Fast green staining, Safranin'O /Fast green staining. The aim is to compare the effectiveness of these staining methods on rat tibia growth plate tissues and to study the different histochemical staining in the growth plate tissue morphogic.Methods: The normal rat tibia tissue samples were fixed, decalcified, dehydrated, and embedded in paraffin. The sections were deparaffinized and rehydrated. (1) HE staining: stained with hematoxylin for 5 minutes; washed with water for 1 minute; step down in 1% hydrochloric acid alcohol for several seconds; stained with eosin for 3 minutes; washed with water for 1 minute; dehydrated; claritied with dimethylbenzene; covered. (2) Masson trichrome staining (compound staining with aniline blue): stained with liquid A for 5 minutes; washed in 0.2% acetic acid; immerged in 5% phosphotungstic acid for 5-10 minutes; washed with 0.2% acetic acid two times; stained with liquid B for 5 minutes; washed with 0.2% acetic acid two times; dehydrated; claritied; covered. Masson trichrome staining (compound staining with light green): stained with liquid A for 5 minutes; washed in 0.2% acetic acid; immerged in 5% phosphotungstic acid 5-10 minutes; washed with 0.2% acetic acid two times; stained with liquid B for 5 minutes; washed with 0.2% acetic acid two times; dehydrated; claritied; covered. (3) AB-PAS staining: washed in distilled water for 1 minute; dipped in 3% acetic acid for 3 minutes; stained with Alcian blue for 30 minutes; dipped in 3% acetic acid for 3 minutes; washed in distilled water for several times; dipped in periodic acid for 10 minutes; washed with water; washed in distilled water for 2 times; stained with Shiff reagent for 10-20 minutes (according to room temperature); washed with water for 2-5 minutes; stained with Harris hematoxylin; differentiated with 0.5% hydrochloric acid alcohol for several seconds; washed with distilled water for several times; dehydrated; claritied; covered. (4) Toludine blue staining: stained with toludine blue solution for 30 minutes; washed with water for 1 minute; differentiated with acetic acid for several seconds; washed with distilled water for 2×2minutes; air-dried; claritied; covered. (5)Safranine staining: stained with 0.1% Tibetan red flower solution for 2-3 minutes; differentiated with acetic acid for several seconds; dehydrated; claritied; covered. (6) AZAN trichrome staining: stained with azocarmin solution for 4-8 hours at 37℃; washed with distilled water for several times; differentiated with 95% alcohol which contains 1% aniline until the tissue showed pale red; stained with 0.5% phosphotungstic acid for 3-5 minutes; stained with aniline blue -chrysoidine solution for 0.5-1 hour; dried with filter paper; differentiated with 95% alcohol for 0.5-1 minute; dehydrated; claritied; covered. (7) Van Gieson staining: stained with Weigert iron hematoxylin solution foor 5-10 minutes; washed with water; stained with Van Gieson solution for 0.5-1 minutes; differentiated with 95% alcohol for several seconds; dehydrated; claritied; covered. (8) Mallory trichrome staining: stained with potassium dichromate solution for 10 minutes; washed with water for 2 minutes; washed with distilled water for 2 times; stained with acidroseine solution for 2 minutes; washed with distilled water; stained with aniline blue solution for 20 minutes; differentiated with 95% alcohol; dehydrated with 100% alcohol; claritied; covered. (9) Safranin'O staining: stained with 1% Safranin'O solution for 3 minutes; differentiated with 95% alcohol; air-dried; claritied; covered. (10) Fast Green staining: stained with 1% Fast Green solution for 3 minutes; differentiated with 95% alcohol; air-dried; claritied; covered. (11) Safranin'O /Fast Green staining: stained with 1% Safranin'O solution for 3 minutes; stained with 1% Fast Green solution for 3 minutes; differentiated with 95% alcohol for several seconds; stained with 1% Safranin'O solution for 2 minutes; differentiated with 95% alcohol for several seconds; air-dried; claritied; covered. After staining, we observe the growth plate tissue with OLIMPUS microscope, and photomicrographs were acquired with a microscope Leica DMR. To compare the effectiveness of these staining methods on rat tibia growth plate tissues.Results: (1) HE staining: the cartilage tissue showed blue in color, we can see the chondrocytes in different zones, the cartilage lacuna displayed no color. Mature bone teabecula displayed pink in color. (2) Masson trichrome staining methods (compound staining with aniline blue): the cartilage tissue displayed pale blue, we can see the cartilage lacuna with no color and the chondrocytes in different zones. The shape of the bone teabecula is irregular. As the bone teabecula grow, some of them showed red in color and the mature bone teabecula displayed dark blue. With Masson trichrome staining osteoid tissue displayed pale blue and mineralized bone showed dark blue in color. Fibrotic tissue showed a striated blue staining . Masson trichrome staining methods (compound staining with light green): the cartilage tissue displayed pale green, we can see the cartilage lacuna with no color and the chondrocytes in different zones. The shape of the bone teabecula is irregular. As the bone teabecula grow, some of them showed red in color and the mature bone teabecula displayed dark green. With Masson trichrome staining osteoid tissue displayed pale green and mineralized bone showed dark green in color. (3) AB-PAS staining: the cartilage extracellular matrix showed blue, the chondrocytes organize in a typical columnwise orientation, the color goes light from the reserve zone to the hypertrophic zone. Ossification center displayed red in color. We can easily distinguish the growth plate from other tissues. (4) Toluidine blue staining: the cartilage extracellular matrix showed even blue, the typical columnwise cartilage cells loomed in the tissue. The nucleolus showed purple. (5) Safranine staining: the cartilage extracellular matrix showed different orange, the color goes light from the reserve zone of the chondrocytes to the hypertrophic zone. The bone teabecula displayed pink in color. The cartilage zone is quite different from the osteal tissue. (6) AZAN staining trichrome methods: cartilage extracellular matrix showed pale blue, we can see the typical columnwise chondrocytes, every zone of the growth plate displayed pale blue. The bone teabecula showed blue in color, we can see the red cells between the bone teabecula displayed orange. (7) Van Gieson staining: the cartilage tissue showed pale red in color and the cartilage cell nucleus displayed darkish. The bone teabecula showed irregular in shape, it displayed red in color. There are palm red cells between the bone teabecula. We can easily distinguish the growth plate from other tissues. (8) Mallory trichrome staining: the cartilage extracellular matrix and the bone teabecula displayed blue, the color of the bone teabecula is the darkest. The chondrocytes'nucleus displayed red in color and the red cells between the bone teabecula displayed orange. (9)Safranin'O staining: the cartilage extracellular matrix and the bone teabecula displayed pink. The color goes light from the chondrocytes reserve zone to the hypertrophic zone and the color of the bone teabecula is the lightest. (10) Fast green staining: the bone teabecula showed pale blue in color, the chondrocyte and the extracellular matrix hardly colored up. (11) Safranin'O/Fast green staining: the cartilage extracellular matrix showed pink in color. The color goes light from the reserve zone to the hypertrophic zone. The bone teabecula displayed green, the red cells between the bone teabecula showed green in color. There are a greet contrast between the cartilage tissue and the osteal tissue.Conclusion: The AB-PAS staining, Van Gieson staining and the Safranin'O/Fast green staining methods are better then other methods in the study of the growth plate histology, they could also be used in the study of bone formation.