ARTICLE |
Correspondence to: Andrew Scutt, Institute of Child Health, Children's Hospital, Western Bank, Sheffield S10 2TH, UK. E-mail: a.m.scutt@sheffield.ac.uk
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Summary |
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We have developed methodology that enables alkaline phosphatase (ALP) to be histochemically stained reproducibly in decalcified paraffin-embedded bone and cartilage of rodents. Proximal tibiae and fourth lumbar vertebrae were fixed in periodatelysineparaformaldehyde (PLP) fixative, decalcified in an EDTA-G solution, and embedded in paraffin. In the articular cartilage of the proximal tibia, ALP activity was localized to the hypertrophic chondrocytes and cartilage matrix of the deep zone and the maturing chondrocytes of the intermediate zone. The cells and matrix in the superficial zone did not exhibit any enzyme activity. In tibial and vertebral growth plates, a progressive increase in ALP expression was seen in chondrocytes and cartilage matrix, with activity being weakest in the proliferative zone, higher in the maturing zone, and highest in the hypertrophic zone. In bone tissue, ALP activity was detected widely in pre-osteoblasts, osteoblasts, lining cells on the surface of trabeculae, some newly embedded osteocytes, endosteal cells, and subperiosteal cells. In areas of new bone formation, ALP activity was detected in osteoid. In the bone marrow, about 20% of bone marrow cells expressed ALP activity. In adult rats, the thickness of the growth plates was less and ALP activity was enhanced in maturing and hypertrophic chondrocytes, cartilage matrix in the hypertrophic zone, and primary spongiosa. This is the first time that ALP activity has been successfully visualized histochemically in decalcified, paraffin-embedded mineralized tissues. This technique should prove to be a very convenient adjunct for studying the behavior of osteoblasts during osteogenesis.
(J Histochem Cytochem 50:333340, 2002)
Key Words: alkaline phosphatase, bone, cartilage, growth plate
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Introduction |
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ALKALINE PHOSPHATASE (ALP) is a membrane-bound metalloenzyme which catalyzes the hydrolysis of phosphomonoesters at an alkaline pH. At least four isozymes of ALP have been identified in humans: nonspecific liver/bone/kidney, intestinal, placental, and germ-cell ALP. The ALP produced by osteoblasts (OBs) and neutrophilic granulocytes is of the nonspecific liver/bone/kidney type ALP. The importance of ALP in bone formation and mineralization was first recognized by
The localization of ALP in calcified tissues presents problems that do not exist with other histochemical methods. Its activity is quite sensitive to fixation and is denatured by moderately high temperatures (
A method has been described by which ALP can be localized in ethanol-fixed undecalcified bone. However, it was remarked that the material had to be processed immediately after fixation and that ALP activity was lost after decalcification (
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Materials and Methods |
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Animals and Harvest of Tissues
Wistar rats (200300 g) or embryonal mice were sacrificed by cervical dislocation according to home office guidelines. The left proximal tibiae and the fourth lumbar vertebral bodies were removed, dissected free of soft tissue, and fixed with PLP fixative (2% paraformaldehyde containing 0.075 M lysine and 0.01 M sodium periodate solution, pH 7.4, stored at 5C) for 24 hr at 4C as previously described (
Decalcification of Tissues
After fixation, the specimens were washed for 12 hr at 4C in each of the following series of solutions: 0.01 M PBS containing 5% glycerol, 0.01 M PBS containing 10% glycerol, and 0.01 M PBS containing 15% glycerol. The specimens were then decalcified in EDTA-G solution (14.5 g EDTA, 1.25 g NaOH, and 15 ml glycerol were dissolved in distilled water and the pH was adjusted to pH 7.3. The solution was then made up to 100 ml and stored at 4C) for 1014 days at 4C as previously described (
Washing of Decalcified Tissues
To remove EDTA and glycerol from the decalcified tissues, they were washed at 5C for 12 hr in successive washes of 15% sucrose and 15% glycerol in PBS, 20% sucrose and 10% glycerol in PBS, 20% sucrose and 5% glycerol in PBS, 20% sucrose in PBS, 10% sucrose in PBS, 5% sucrose in PBS, and 100% PBS as previously described (
Dehydration and Paraffin Embedding
Tissues were dehydrated in a graded series of alcohols and embedded in low-melting-point paraffin using a Shandon Citadel 2000 automatic tissue processor (Shandon Scientific; Runcorn, UK).
Tissue Sectioning
The tissue blocks were trimmed down to the tissue surface. The block was then placed in a microtome and 5-µm sections were cut at frontal for tibiae and at sagittal for vertebrae. Tissue sections were floated in a water bath at 48C and collected on poly-L-lysine-coated glass slides. Sections were stained with hematoxylin and eosin (HE) and histochemically for ALP.
Histochemical Demonstration of ALP Activity in Paraffin Sections
ALP histochemistry was performed following a modified version of a previously described method (
Preparation of Rat Bone Marrow Cells (BMCs) and Cytospin Preparations
Tibiae and femurs of 200-g male Wistar rats were removed under aseptic conditions and the BMCs were flushed out with standard medium. The cells were dispersed by repeated pipetting and a single-cell suspension was achieved by forcefully expelling the cells through a 21-gauge syringe needle. One-ml cell suspensions were centrifuged at 3000 rpm for 5 min. The supernatant was aspirated off and the pellet was fixed with 1 ml 10% neutral phosphate-buffered formalin or PLP fixative for 20 min and resuspended. Cell suspensions of 150 µl were cytospun onto slides at 500 rpm for 5 min. After air-drying, the slides were stored at -20C until staining.
Cytochemical Demonstration of ALP Activity in BMCs
ALP cytochemistry is much easier than ALP histochemistry because the cells do not undergo a long stringent process of decalcification and paraffin embedding. The cells were cytospun onto slides and incubated for 15 min at RT in the ALP substrate solution as described above. After washing with distilled water, the cells were counterstained with Vector methyl green nuclear counterstain and mounted with Kaiser's glycerol jelly. After staining, the numbers of total cells with nuclei, ALP strong positive cells, and ALP weak positive cells within five fields of view (x400) were recorded. The percentages of ALP strong and weak positive cells were calculated and the results are expressed as means ± SD.
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Results |
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Effect of Fixative on ALP Activity in BMC Cytospin Preparations
In a preliminary experiment using cytospin preparations of whole BMCs, the effects of fixation with either 10% neutral phosphate-buffered formalin or PLP fixative were compared. It was found that ALP activity in BMCs was well preserved after fixation with PLP fixative (Fig 3G) but was totally absent after fixation with 10% neutral phosphate-buffered formalin (results not shown). In the cytospin preparations the BMCs could be divided into strong and weakly positive cells according to the intensity of ALP staining (Fig 3H). The percentage of ALP strong and weak positive BMCs was 4.4 ± 0.2% and 16.5 ± 0.4% of total BMCs, respectively. It was also found that after fixation with PLP fixative, the cytospin preparations of BMC could be stored at -20C for up to 2 years and still retain their ALP activity.
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Histochemical Demonstration and Localization of ALP Activity in Decalcified, Paraffin-embedded Bone and Cartilage Sections
Following on from the above experiment using cytospin preparations of whole BMCs, tibiae were fixed with either 10% neutral phosphate-buffered formalin or PLP fixative, decalcified, paraffin-embedded, and then sections were stained using an ALP protocol in which Mg2+ ions, lost during the decalcification process were replaced by preincubation with MgCl2. As before, those fixed with 10% neutral phosphate-buffered formalin were totally devoid of ALP activity in both the growth plate and the primary spongiosa (Fig 1). In contrast, those fixed with PLP successfully retained their ALP activity (see below).
In the articular cartilage of proximal tibiae from 200-g rats, ALP activity was localized to hypertrophic chondrocytes and cartilage matrix of the deep zone and maturing chondrocytes in the intermediate zone. Resting chondrocytes and matrix of superficial zone and the matrix of intermediate zone did not exhibit any enzyme activity (Fig 2A). In older 285-g rats, ALP activity was stronger in hypertrophic chondrocytes and cartilage matrix of the deep zone. However, the distribution of ALP activity in articular cartilage was similar to that of the 200-g rats (Fig 2B).
In 200-g rat tibial and vertebral growth plates, a progressive increase in ALP expression was seen in chondrocytes and cartilage matrix, with activity being weakest in the proliferative zone, higher in the maturing zone, and highest in the hypertrophic zone. No ALP activity was detected in resting chondrocytes and the matrix of the reserve zone (Fig 2C2G). In 285-g rats, the growth plates were narrower and ALP activity was stronger in maturing and hypertrophic chondrocytes, the cartilage matrix of the hypertrophic zone, and the primary spongiosa, compared to that of 200-g rats.
In bone, ALP activity was detected widely in pre-osteoblasts, osteoblasts, lining cells on the surface of trabeculae, some newly embedded osteocytes, endosteal cells, and subperiosteal cells (Fig 3A3F). In areas of new bone formation, ALP activity was detected in the osteoid (Fig 3D). However, no ALP activity could be dectected in deeply embedded osteocytes and calcified bone matrix. In general, ALP activity seen in bones from older 285-g rats was stronger compared to that of 200-g rats.
Expression of ALP in Z/AP and Z/AP;Creactin Mice
To further study the utility of this method, ALP expression was investigated in tibiae and vertebrae from Z/AP and Z/AP;Creactin mouse embryos. Z/AP transgenic mice express ubiquitously the lacZ reporter gene under the control of the CMV enhancer and the chicken ß-actin promoter (
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Reproducibility of the Histochemical Technique for ALP
To examine the reproducibility of the histochemical technique for ALP developed in this study, specimens were harvested and processed at different time points and stained simultaneously, or the sections from the same rats were stained repeatedly three times. ALP activity was demonstrated very well in all specimens harvested at different time points. When we stained repeatedly for three times in tibial and vertebral sections from 45 rats, ALP activity in the same section was demonstrated at the same levels.
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Discussion |
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So far as we are aware, this is first time that ALP activity has been demonstrated histochemically in PLP-fixed, EDTA-G decalcified, paraffin-embedded mineralized tissues. This histochemical technique for ALP was found to be highly reproducible and can be performed on large numbers of blocks in conventional histology laboratories. This is a significant advantage over frozen or plastic-embedded samples because paraffin-embedded bone can be used to successfully localize many other enzymes, antigens, RNA, and DNA and this development means that these can now be co-localized with ALP, a central enzyme in bone metabolism. This technique would therefore be ideal for studying osteogenesis in vivo.
Fixation was found to be a critical step in the successful demonstration of ALP activity in decalcified paraffin-embedded tissues. Our results show that the use of neutral phosphate-buffered formalin destroys all ALP activity, whereas PLP fixation preserves ALP activity very well, particularly after long-term fixation of larger tissue blocks. The ALP activity was still well preserved in BMCs stored at -20C for up to 2 years after fixation with PLP. Although ALP activity is preserved using other fixatives and can subsequently be demonstrated in plastic-embedded, decalcified frozen and undecalcified paraffin-embedded tissues (
The replacement of magnesium chloride leached out during the decalcification process was also found to be crucial for the demonstration of ALP activity in decalcified mineralized tissues. To achieve this, the sections were preincubated in 1% magnesium chloride in 100 mM Tris-maleate buffer before staining. Omission of this step results in total lack of staining for ALP. This is consistent with the work of
By employing the histochemical technique for ALP established in this study, the localization of ALP on bone and cartilage was investigated in rats of different ages. The distribution of ALP activity as assessed in the present study in decalcified paraffin-embedded bone and cartilage matches that as assessed in decalcified frozen bone and cartilage (
Tissue architecture was well preserved when whole proximal ends of tibiae and vertebral bodies were processed, which allowed the observation of several areas of cartilage and bone simultaneously. ALP activity was evident not only in the cartilage of growth plates, osteoblasts, endosteal cells, and bone marrow cells, as described previously, but also in periosteal cells and articular cartilage. Many subperiosteal cells expressed ALP, indicating that these cells may be osteoblast precursors. ALP was localized in the same cell type in articular cartilage as that in growth plate. However, less ALP was detected in the matrix of deep articular cartilage than in the matrix of the growth plate hypertrophic zone. We also demonstrated that ALP activity was detectable in the osteoid of areas of new bone formation but not in calcified bone matrix.
ALP activity can also be localized to large numbers of bone marrow cells. Because it is now believed that osteoblast precursors reside in the stromal compartment of the bone marrow (
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Acknowledgments |
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Supported by Schering AG, Berlin, and by Research into Ageing.
Received for publication June 20, 2001; accepted October 24, 2001.
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