TY - JOUR
T1 - Overview and assessment of the histochemical methods and reagents for the detection of β-galactosidase activity in transgenic animals
AU - Trifonov, Stefan
AU - Yamashita, Yuji
AU - Kase, Masahiko
AU - Maruyama, Masato
AU - Sugimoto, Tetsuo
N1 - Funding Information:
The purpose of this review is to provide an overview of the current histochemical methods for detecting β-galactosidase (lacZ gene of Escherichia coli) expression in genetically engineered animals. Based on a comparison of their sensitivity and specificity, we are proposing an optimized and enhanced method for β-galactosidase detection in histological sections of the transgenic mouse brain. The experiments were carried out with 10-week-old male heterozygous B6.Cg-Tg(Nes-cre)1Nogu mice (No. RBRC02412), provided by RIKEN BioResource Center (BRC) through the National BioResource Project of the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan. These mice were chosen over the ubiquitously β-galactosidase-expressing Rosa26 mice because the level of expression of the lacZ gene is lower, which makes them suitable for a comparison of the sensitivity of different substrates. These transgenic mice express Cre recombinase under the control of the Nestin promoter/enhancer. The Nestin-Cre transgene contains rat nestin promoter, Cre recombinase gene and IRES (internal ribosomal entry site)-lacZ-polyA (Mishina and Sakimura ; Tanaka et al. ). C57BL/6J wild-type male mice of the same age were used as controls. All experiments were performed in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised 1996) and the Kansai Medical University local guidelines for animal experimentation (issued 9 March 1999; registration number of the current research proposal, 12-064; permit number, 25-055). The study had the full approval of the Institutional Committee for Animal Experimentation. All efforts were made to reduce the number of animals and their suffering. The animals were deeply anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and transcardially perfused with 0.9 % saline solution (1 ml/g b.wt.), followed by freshly prepared 4 % paraformaldehyde, 1.25 mM EGTA and 2 mM MgCl in 0.1 M phosphate buffer (PB, pH 7.5; 2 ml/g b.wt.). The brains were dissected, postfixed by immersion in the same fixative for 2 h at 4 °C, and then transferred to 30 % sucrose in 0.1 M PB (pH 7.5) at 4 °C until they sank. The brains were then frozen on a sliding microtome, and 40-µm-thick sections were cut on the coronal plane. The tissue sections were collected in cryoprotection buffer (30 % sucrose, 30 % ethylene glycol, 50 mM PB) and stored at −20 °C until processing. They were then washed three times in rinse solution containing 0.02 % Nonidet P-40, 0.01 % sodium deoxycholate, 2 mM MgCl, 1.25 mM EGTA and 0.1 M phosphate buffer (pH 7.5) for 15 min each. The staining solution consisted of either 1 mg/ml X-gal (Nacalai Tesque, Inc., Kyoto, Japan) or 1 mg/ml Salmon-β-D-gal (Biosynth International, Inc., Itasca, IL, USA), in combination with (1) 5 mM KFe(CN) and 5 mM KFe(CN), (2) 0.4 mg/ml NBT (Roche Diagnostics, Mannheim, Germany), (3) 0.2 mg/ml TNBT (Sigma-Aldrich, St. Louis, MO, USA), or (4) 0.8 mg/ml INT (Tokyo Chemical Industry Co., Ltd, Tokyo, Japan) respectively, in rinse solution at 37 °C, protected from light. Tetrazolium salts were dissolved in 70 % dimethylformamide. The staining reaction was monitored, and the optimal duration was determined for every combination of substrates. Finally, tissue sections were washed in 0.1 M phosphate-buffered saline (PBS), mounted on gelatin-coated slides, dehydrated with successive changes of ethanol (50, 75, 95, 100 and 100 % ethanol), clarified in three changes of xylene, and sealed with Canada balsam and a coverslip. The sections stained with a combination of X-gal or Salmon-gal and INT were washed in 0.1 M PBS, mounted on gelatin-coated slides and sealed with CC/Mount (Diagnostic Biosystems, Pleasanton, CA, USA) and a coverslip, thus avoiding dehydration and clearing in xylene, as INT precipitate dissolves completely in these organic solvents. Adjacent coronal sections were stained with cresyl violet to facilitate identification of nuclei and fiber tracts. The stained sections were viewed under a Nikon Eclipse E800 light microscope (Nikon Corp., Tokyo, Japan) under bright-field illumination. The nomenclature of nuclei and related fiber tracts was adopted from Paxinos and Franklin () (Fig. a, b). 2 ® 2 3 6 4 6
Funding Information:
The authors thank Fumio Yamashita and Tetsuji Yamamoto for technical assistance, and Yuki Okada for expert secretarial work. This work is supported in part by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and the Science Research Promotion Fund of the Japan Private School Promotion Foundation.
Publisher Copyright:
© 2015, The Author(s).
PY - 2016/1/1
Y1 - 2016/1/1
N2 - Bacterial β-galactosidase is one of the most widely used reporter genes in experiments involving transgenic and knockout animals. In this review we discuss the current histochemical methods and available reagents to detect β-galactosidase activity. Different substrates are available, but the most commonly used is X-gal in combination with potassium ferri- and ferro-cyanide. The reaction produces a characteristic blue precipitate in the cells expressing β-galactosidase, and despite its efficiency in staining whole embryos, its detection on thin tissue sections is difficult. Salmon-gal is another substrate, which in combination with ferric and ferrous ions gives a reddish-pink precipitate. Its sensitivity for staining tissue sections is similar to that of X-gal. Combining X-gal or Salmon-gal with tetrazolium salts provides a faster and more sensitive reaction than traditional β-galactosidase histochemistry. Here, we compare the traditional β-galactosidase assay and the combination of X-gal or Salmon-gal with three tetrazolium salts: nitroblue tetrazolium, tetranitroblue tetrazolium and iodonitrotetrazolium. Based on an assessment of the sensitivity and specificity of the different combinations of substrates, we are proposing an optimized and enhanced method for β-galactosidase detection in histological sections of the transgenic mouse brain. Optimal staining was obtained with X-gal in combination with nitroblue tetrazolium, which provides a faster and more specific staining than the traditional X-gal combination with potassium ferri- and ferro-cyanide. We recommend the X-gal/nitroblue tetrazolium staining mixture as the first choice for the detection of β-galactosidase activity on histological sections. When faster results are needed, Salmon-gal/nitroblue tetrazolium should be considered as an alternative, while maintaining acceptable levels of noise.
AB - Bacterial β-galactosidase is one of the most widely used reporter genes in experiments involving transgenic and knockout animals. In this review we discuss the current histochemical methods and available reagents to detect β-galactosidase activity. Different substrates are available, but the most commonly used is X-gal in combination with potassium ferri- and ferro-cyanide. The reaction produces a characteristic blue precipitate in the cells expressing β-galactosidase, and despite its efficiency in staining whole embryos, its detection on thin tissue sections is difficult. Salmon-gal is another substrate, which in combination with ferric and ferrous ions gives a reddish-pink precipitate. Its sensitivity for staining tissue sections is similar to that of X-gal. Combining X-gal or Salmon-gal with tetrazolium salts provides a faster and more sensitive reaction than traditional β-galactosidase histochemistry. Here, we compare the traditional β-galactosidase assay and the combination of X-gal or Salmon-gal with three tetrazolium salts: nitroblue tetrazolium, tetranitroblue tetrazolium and iodonitrotetrazolium. Based on an assessment of the sensitivity and specificity of the different combinations of substrates, we are proposing an optimized and enhanced method for β-galactosidase detection in histological sections of the transgenic mouse brain. Optimal staining was obtained with X-gal in combination with nitroblue tetrazolium, which provides a faster and more specific staining than the traditional X-gal combination with potassium ferri- and ferro-cyanide. We recommend the X-gal/nitroblue tetrazolium staining mixture as the first choice for the detection of β-galactosidase activity on histological sections. When faster results are needed, Salmon-gal/nitroblue tetrazolium should be considered as an alternative, while maintaining acceptable levels of noise.
KW - Nitroblue tetrazolium
KW - Salmon-gal
KW - X-gal
KW - lacZ
KW - β-galactosidase
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U2 - 10.1007/s12565-015-0300-3
DO - 10.1007/s12565-015-0300-3
M3 - Article
C2 - 26394634
AN - SCOPUS:84951906633
SN - 1447-6959
VL - 91
SP - 56
EP - 67
JO - Anatomical Science International
JF - Anatomical Science International
IS - 1
ER -