Improved methods for detection of β-galactosidase (lacZ) activity in hard tissue

The β-galactosidase gene (lacZ) of Escherichia coli is widely used as a reporter gene. The expression of lacZ can be detected by enzyme-based histochemical staining using chromogenic substrates such as 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-gal). Because the enzymatic activity of lacZ is vulnerable to high temperatures and acid treatment for demineralization, detection of lacZ on paraffinized sections is difficult, especially for hard tissues, which require demineralization before sectioning in paraffin. To circumvent this problem, whole-mount X-gal staining before sectioning is performed. However, detection of lacZ activity in the center of larger portions of hard whole adult tissues is challenging. In this study, focusing on fixation procedures, we determined the conditions conducive to improved detection of lacZ activity in deeper areas of whole tissues. We used an annexin a5 (Anxa5)-lacZ reporter mouse model in which the Anxa5 expression in hard tissue is indicated by lacZ activity. We found that lacZ activity could be detected throughout the periodontal ligament of adult mice when fixed in 100% acetone, whereas it was not detected in the periodontal ligament around the root apex fixed in glutaraldehyde and paraformaldehyde. This staining could not be detected in wild-type mice. Acetone maintains the lacZ activity within 48 h of fixation at both 4°C and at room temperature. In conclusion, acetone is the optimal fixative to improve permeability for staining of lacZ activity in large volumes of adult hard tissues.


Introduction
Genetically engineered mice are used to elucidate molecular functions of genes in vivo. In such animals, in situ biochemical assays assist in the detection of temporal and spatial expression patterns for engineered genes and proteins (e.g. Sanes et al. 1986, Houzelstein et al. 1997Diamond et al. 2000;Lézot et al. 2000). In addition, cells labeled by engineered genes are used to distinguish between donor and recipient cells in cell tracing studies (e.g. Sanchez-Ramos et al. 2000;Lekic et al. 2001). The -galactosidase gene (lacZ) of Escherichia coli is often used as a reporter gene for these purposes (Cui et al. 1994).
-Galactosidase hydrolyzes X-gal to form a highly sensitive blue precipitate. When histochemical staining is performed on the paraYnized section, the enzymatic activity is degraded by the high temperatures applied during the process of tissue preparation. The lacZ activity is lost under incubation at temperatures above 50°C, but not below 42°C (Avé et al. 1997;Cho et al. 2010). Moreover, in sections of hard tissues such as tooth and/or bone, detection of lacZ is often diYcult because these tissues require complete decalciWcation (demineralization) to prepare appropriate sections for histological analysis. Because acid demineralization agents tend to weaken lacZ activity, demineralization by ethylene diamine tetra-acetic acid (EDTA) and detection using frozen sections is performed for teeth, although the resolution of frozen sections is lower than that of paraYnized sections (Cho et al. 2010). To prevent these problems, tissues and embryos are stained by whole-mount X-gal staining before sectioning. Conditions for detection of lacZ activity in diVerent Wxatives and stains have been previously investigated, and the standard protocols have been established (Murti and Schimenti 1991;Sanchez-Ramos et al. 2000;Pereira 2001;Gioglio et al. 2002;Ma et al. 2002;Takahashi et al. 2003;Bell et al. 2005;Bolon 2008). Despite these protocols, it is diYcult to speciWcally detect the lacZ activity in the deeper regions of larger tissues, especially in hard tissues. An appropriate protocol for detection of lacZ activity in whole hard tissues has not yet been established.
In this study, we determined the conditions conducive to improved detection of lacZ activity in deeper areas of hard tissues. We used an annexin a5 (Anxa5)-lacZ reporter mouse model (Brachvogel et al. 2001;Brachvogel et al. 2005), in which the Anxa5-lacZ gene is expressed in the periodontal ligament tissue between the alveolar bone and the teeth (Shimada et al. in preparation).

Materials and methods
All animal experiments were approved by the Institutional Animal Care Committee and the Recombination DNA Experiment and Biosafety Committee of the Tsurumi University School of Dental Medicine.

Tissue preparation
A total of 44 Anxa5-lacZ reporter mice (Brachvogel et al. 2001) aged 3-11 months were used. As a negative control, 12 wild-type C57BL/6J mice (CLEA Japan, Inc., Tokyo, Japan) of similar ages were used. Mice were killed by cervical dislocation. The composition of Wxatives and the Wxation conditions are shown in Table 1. In the preliminary study, we identiWed Anxa5 expression in periodontal tissues. To conWrm Anxa5-lacZ expression in periodontal tissues, extracted mandibular Wrst molars with the adherent periodontal ligament were Wxed in 100% acetone and 0.2% glutaraldehyde. For whole-mount X-gal staining in larger hard tissue, the maxillae of adult mice were examined. The maxillae were skinned, dissected into approximately 20mm tall, 15-mm wide, and 10-mm thick sections, and Wxed in acetone, glutaraldehyde, and paraformaldehyde (PFA). To test the tissue permeability of Wxatives, the maxillae were Wxed in acetone, glutaraldehyde, and PFA with or without removal of palatine mucosa before Wxation. The other tissues were Wxed in glutaraldehyde and irradiated in a microwave (MW) oven (glutaraldehyde-MW, Murti and Schimenti 1991).
Staining of lacZ activity, demineralization, and preparation of paraYnized sections Fixed tissues were washed with a reaction buVer consisting of 100 mM sodium phosphate buVer (pH 7.3), 2 mM magnesium chloride, 0.02% Nonidet P-40, and 0.01% sodium deoxycholate. The tissues were then stained for lacZ activity with a reaction buVer containing 1 mg/ml X-gal, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 20 mM Tris-hydrochloric acid (pH 7.3). After staining, tissues were post-Wxed in 4% PFA for 24 h at 4°C and demineralized with Morse's solution (22.5% formic acid and 10% sodium citrate) for 36 h. Demineralized specimens were dehydrated in graded ethanol, cleared in Tissue-Tek ® Tissue-Clear™ (Sakura Finetek Japan, Tokyo, Japan), and embedded in Tissue-Tek ® Xpress ® ParaYn Wax II60 (Sakura Finetek Japan, Tokyo, Japan). Sagittal sections of maxillary Wrst molars, with a thickness of 6 m, were placed on slides pre-coated with aminopropyltriethoxysilane (Matsunami glass, Osaka, Japan). Assessment of Anxa5-lacZ detection Teeth that were stained after extraction were photographed using a digital camera (DP71, Olympus, Tokyo, Japan) connected to a stereomicroscope (MZFLIII, Leica Microsystems Japan, Tokyo, Japan). Stained sections were photographed using a digital camera connected to a diVerential interference microscope (Eclipse 80i, Nikon, Tokyo, Japan). The area stained with X-gal in rectangular area measuring 65 m wide and 380 m tall including periodontal ligament in mesial side of mesial root and distal side of distal root was measured by ImageJ 1.39 (NIH, USA) (Fig. 2e). The stained area was calculated as percent area against total area and the diVerence of percent area among Wxation conditions were statistically analyzed by ScheVe's test. The Wxation condition which shows the most extensive X-gal staining was assessed as optimal.

Results
Whole-mount lacZ staining of the periodontal tissue attached to an extracted tooth In our preliminary experiment, we observed Anxa5 expression in the periodontal tissue. Using Anxa5-lacZ knock-in mice, we extracted Wrst molars of the mandible and examined lacZ expression as whole mounts. Extracted teeth were Wxed in glutaraldehyde or acetone for 8 h at 4°C and whole teeth containing periodontal ligament tissues were stained with X-gal. Periodontal ligaments attached to tooth roots from Anxa5-lacZ mice showed strong blue staining (Supplemental Fig. 1c, d). This was not observed in the wild-type C57BL/6J mice (Supplemental Fig. 1a, b). The distribution of blue staining by X-gal was the same under both sets of Wxation conditions. These results indicate that lacZ is uniformly stained under both conditions when tissues were directly exposed to Wxative reagents.
Optimal conditions for Wxation for whole-mount lacZ staining of the maxillae We next examined if lacZ expression in the periodontal ligament could be detected when we stained maxillary bone and teeth as whole maxillary tissues. Maxillae from Anxa5-lacZ mice were Wxed under various Wxative conditions (Table 1), stained with X-gal, postWxed in PFA, demineralized with Morse's solution, and embedded in paraYn, according to the conventional procedure. In the PFA (Fig. 1b) and glutaraldehyde (Fig. 1c) Wxed tissues, lacZ activity could be detected in palatine mucosa, gingival tissue, and the periodontal tissues only around the cervical area, and not around the root apices. In contrast, lacZ activ-ity could be detected in all over the periodontal ligament tissues and apical part of dental pulp Wxed with acetone (Fig. 1a). The mean percent area of X-gal staining in the specimen Wxed with acetone was more than twice as large as those with other Wxatives (Fig. 1e, p < 0.01). In the wildtype mice (Fig. 1d), blue staining of X-gal was not observed, indicating that endogenous -galactosidase activity does not give rise to blue staining under our experimental conditions. These results suggest that acetone is the most eVective Wxative for detecting lacZ activity in deeper areas of maxillary tissue.
Permeability of Wxatives aVects whole-mount staining of lacZ When we stained whole maxillary tissue, the stained regions diVered according to the Wxative used. We considered that the diVerences in staining among the diVerent Wxatives were due to their diVerent tissue permeabilities. To clarify this, we experimentally removed the physical barrier of the maxillae, i.e., mucosal epithelium, to increase the permeability of the Wxatives. When maxillae were Wxed in PFA (Supplemental Fig. 2b) and glutaraldehyde (Supplemental Fig. 2c) after removal of the palatine mucosa, the lacZ activity could be detected extensively in the deeper areas of the periodontal tissues relative to the tissues treated without removal of the mucosa (Fig. 1b, c, respectively). In contrast, the tissues Wxed in acetone did not show a signiWcant diVerence of area stained with X-gal before or after removal of the mucosa (Fig. 1a, Supplemental Fig. 2a). Irrespective of Wxation conditions, intensity of X-gal staining in the periodontal ligament around cervical area increased after removal of palatine mucosa compared with opposite side of maxillae without removal of palatine mucosa. This increased lacZ activity could be due to the increased expression of annexin a5 protein, which was con-Wrmed by immunohistochemical detection using annexin V antibody (Supplemental Fig. 3). When maxillae were Wxed in glutaraldehyde and irradiated in the microwave oven without removal of the palatine mucosa (Supplemental Fig. 2d), lacZ activity was detected in the periodontal tissues around root apices, although the signal was relatively weak in deeper areas. When maxillae were Wxed in glutaraldehyde for a longer time, the X-gal staining was expanded to deeper areas in the periodontal ligament (Fig. 2b, c). Taken together, these results indicate that the improved permeability induced by the Wxatives allows us to detect lacZ activity in deeper areas of the periodontal ligament.
In general, the permeability of Wxatives into tissue becomes lower at low temperatures, whereas enzymatic activity can be eVectively retained at low temperatures. In contrast, excessive Wxation conditions, such as the use of higher concentrations of Wxatives and/or Wxation for a longer time, may cause loss of enzymatic activity. Therefore, we sought to determine the optimal time and temperature conditions by varying these parameters for the process of Wxation using acetone. When maxillae were Wxed in acetone for 8, 24, and 48 h at 4°C (Fig. 2 g-i), lacZ activities detected in the periodontal tissues were found to be similar to those measured at room temperature (Fig. 2 d-f). The diVerent Wxation times ranging from 8 to 48 h did not produce a signiWcant diVerence in lacZ activity with acetone ( Fig. 2 d-i), but the maxillae Wxed in acetone had signiWcantly higher lacZ activity than maxillae Wxed in glutaraldehyde under the same conditions of time and temperature (Fig. 2 a-c).
As a result, Anxa5-lacZ expressing cells could be clearly detected in the periodontal ligament around the root apex of tissues Wxed in acetone for 8 h at 4°C (Supplemental  Fig. 4b). Strong Anxa5-lacZ signals were also observed on the surface of alveolar bone (Supplemental Fig. 4b).

Discussion
Various improved methods for histological observation of lacZ-expressing cells have been reported, including (a) staining with chromogenic substrates after sectioning u n d e r m ai n te n an ce o f l a cZ e n z y m at i c ac t i v i t y , (b) immunohistological detection of lacZ antigen after sectioning, and (c) sectioning after staining of lacZ activity with chromogenic substrates. Because demineralization using acid and paraYnization at high temperature causes loss of lacZ enzymatic activity, detection of lacZ activity speciWcally in hard tissue requires additional improvements to these protocols. As the Wrst method, Avé et al. (1997) succeeded in detecting lacZ activity in a paraYnized section of an embryo using low-melting point paraYn at 42°C. This method was less sensitive at 56°C. Cho et al. (2010) reported an improved method for rapid demineralization using 0.1 M EDTA at 42°C without loss of lacZ activity in frozen sections of hard tissues. The time required for complete demineralization of the skull of a 3-month-old mouse was 5-6 days in this protocol. The low-melting point paraYn, frozen sectioning, and demineralization at higher temperature have the disadvantage of requiring thin sectioning and tissue preservation for higher resolution.
As the second method, Pereira et al. (2006) reported an almost twofold increase in lacZ-transfected dermal cell staining using immunohistochemistry relative to histochemical staining with Bluo-Gal. This method includes activation of antigenicity by procedures such as proteinase pretreatment and heating of the section on the glass slide. In cases of analysis of hard tissue, heating causes speciWc tissue damage to tooth dentine and the surrounding tissue (Shimada et al. 2008). It should be noted that certain antibodies react not only with bacterial -galactosidase but also with endogenous -galactosidase (Kurz et al. 2000). Histochemical lacZ staining can distinguish between two enzymes by diVerences in optimal pH (Bolon 2008).
We chose the third method to detect lacZ activity using whole-tissue staining. For this purpose, appropriate Wxatives have been examined previously. Gioglio et al. (2002) reported that muscle tissue Wxed in a mixture of 2.5% glutaraldehyde and 1% paraformaldehyde showed the highest lacZ activity. Ma et al. (2002) reported that 0.2% glutaraldehyde produced the most consistent and reliable results. Bolon (2008) compared Wxation conditions, including content, concentration, time and temperature for whole tissues, including bone dissected from Rosa26 mice, which have a lacZ-containing transgene, and reported that Wxation in 3% paraformaldehyde for 3 h at 4°C was optimal. Hannouche et al. (2007) reported that Wxation using a mixture of 2% formaldehyde and 0.2% glutaraldehyde for 4 days at 4°C gave the best results for X-gal staining of both cell cultures and bone implanted with mesenchymal stem cells from a vimentin-lacZ knock-in mouse.
In this study, improvement of permeability with PFA and glutaraldehyde by experimental removal of mucosa allowed us to detect the lacZ activity in deeper areas of the periodontal tissue (Supplemental Fig. 2). Interestingly, increased expression of Anxa5 around cervical area of periodontal ligament after invasive removal of palatine mucosa could be detected by both X-gal staining (Supplemental Fig. 2a-c) and immunohistochemistry (Supplemental Fig. 3), although the mechanisms of increased expression of annexin a5 after removal of tissue are unknown. However, detection of lacZ activity in apical region of periodontal ligament was insuYcient by Wxation with PFA and glutaraldehyde. Improvement of detection of lacZ activity in deeper area of the periodontal tissues could be also observed by microwave irradiation (Supplemental Fig. 2d). These results suggest that permeable Wxatives are eVective for staining large whole tissues and are suitable for the hard tissue that is diYcult to micro-dissect before demineralization. Takahashi et al. (2003) showed that 100% acetone produces stronger X-gal staining than 0.2% glutaraldehyde for Wxation of frozen sections of rat skeletal muscle. Based on this report, we compared acetone with other Wxatives and found the most optimal Wxatives for detection of lacZ activity in deeper areas of the tissue. The staining would not be caused by endogenous -galactosidase activity, such as osteoclasts (Odgren et al. 2006), because such staining was not detected in wild-type C57BL/6J mice. Moreover, the immunohistological distribution of annexin a5 protein showed similar results (Shimada et al., in preparation). Acetone could easily permeabilize the tissues even at lower temperatures. This suggests that this Wxative is eVective for retaining the antigenicity for multistaining in the immunohistological study. In fact, we detected injected bromodeoxyuridine by immunohistochemistry after staining of lacZ activity (Shimada et al., in preparation). In other cases, it might be possible that acetone causes tissue shrinkage and hardening or/and loss of antigenicity due to its lipid-and glycolipd-solubility. It has been reported that over-Wxation causes a decrease in lacZ activity (Gioglio et al. 2002;Ma et al. 2002). However, acetone Wxation within 48 h did not decrease the lacZ activity (Fig. 2), suggesting it is a milder Wxative. Acetone must be exchanged frequently to complete the Wxation process. We suggest that incomplete Wxation caused diVuse staining with X-gal (data not shown). It also needs to be careful for staining speciWcity in the keratinized part of the mucosa after Wxation with acetone, although the distribution of annexin a5 could be detected in the keratinized part of palatine mucosa by immunohistochemical detection after Wxation with PFA (Supplemental Fig. 3a).
In conclusion, permeable acetone is the optimal Wxative for staining of lacZ activity in deep areas of whole hard tissues.