ARTICLE |
Correspondence to: Naohiro Tomita, Dept. of Surgery II, Osaka U. Medical School, 2-2 Yamada-oka, Suita City, Osaka 565, Japan.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We describe and discuss a method of protein extraction for Western blot analysis from formalin-fixed, paraffin-embedded tissue sections. From 5-mm2 50-µm-thick tissue sections, an abundance of proteins could be extracted by incubating the sections in lysis buffer containing 2% sodium dodecyl sulfate (SDS) at 100C for 20 min followed by incubation at 60C for 2 hr. Extracts yielded discernible protein bands ranging from 10 kD to 120 kD as identified by SDS-polyacrylamide gel electrophoresis (PAGE). Western blot analysis successfully detected membrane-bound protein such as E-cadherin, cytosolic protein such as ß-catenin, and nuclear proteins including proliferating cell nuclear antigen (PCNA), mutant-type p53, cyclin D1, cyclin E, and cyclin-dependent kinases (CDKs). With this technique, we could examine cyclin D1 and CDK2 expression in small adenomas compared with cancer tissues and normal mucosa. The simple method of protein extraction described here should make it possible to use large-scale archives of formalin-fixed, paraffin-embedded samples for Western blot analysis, and its application could lead to detailed analysis of protein expression. This new technique should yield valuable information for molecular biology. (J Histochem Cytochem 46:397403, 1998)
Key Words: paraffin-embedded tissue, protein extraction, Western blot analysis
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recent advances in molecular technology have identified many genes responsible for or associated with malignant transformation of the cell (
Immunohistochemistry and Western blot analysis are therefore complementary techniques for analysis of protein expression. These two methods, however, can not be applied to the same materials because immunohistochemistry requires processing of the tissues by fixatives such as formalin, which is believed to destroy the proteins and to make it impossible to analyze the same tissues by Western blotting (
In this article we describe a method of protein extraction for Western blot analysis from formalin-fixed, paraffin-embedded tissue sections. Extraction efficiency is comparable to that from frozen tissue samples, and membrane-bound, cytosolic, and nuclear proteins were clearly identified by Western blotting. The method described here should make it possible to use large-scale archives of pathological specimens processed through routine fixation not only for immunohistochemistry but also for Western blot analysis, and can be expected to yield valuable information for molecular biology.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissue Samples
Fresh specimens of colorectal cancer were surgically obtained at the Second Department of Surgery of Osaka University Hospital. Cancer tissues were cut into 5-mm-thick slices and immediately fixed in 10% buffered formalin at 4C for 2448 hr. Four colorectal adenomas were also obtained at surgery together with the cancer tissues. The size of the adenomas ranged from 5 mm to 14 mm maximal diameter (mean 8.8 mm), and all adenomas exhibited moderate dysplasia. These adenomas were fixed in the same manner as used for cancer tissues. The cancer tissues and adenomas were then washed in water for 1 hr and dehydrated in graded ethanol (60%, 80%, 90%, 95%, and 100%) at 4C. Finally, after permeation in xylene, they were embedded in paraffin. The storage period for paraffin blocks used in this study was from 2 months to several years. The full depth of the colorectal wall containing normal mucosa remote from cancer tissues was also cut into 5-mm-thick and 5-cm-long slices and was fixed in buffered formalin at 4C for 2448 h, then embedded in paraffin with the same procedure as used for cancer tissues. Fresh cancer tissues were also collected and stored at -80C until use for protein assay and Western blotting.
Antibodies
Mouse monoclonal antibodies (MAbs) against PCNA (PC-10, IgG2a) and p53 (1801, IgG1) were purchased from Novocastra Laboratories (Newcastle, UK). A mouse MAb against cyclin E (HE-12, IgG1) was acquired from Oncogene Science (Cambridge, MA). The mouse MAb against ß-catenin (IgG1), which was raised against a 23-kD protein fragment corresponding to amino acid residues 571781 of mouse ß-catenin (
Protein Extraction
Paraffin sections 4 µm thick were cut for hematoxylineosin staining and 50-µm-thick serial sections were cut for protein extraction and mounted on plain glass slides. Three 50-µm-thick sections for protein extraction were deparaffinized in xylene, rehydrated in graded ethanol, immersed in distilled water, and air-dried. To exclusively collect 5-mm2 cancer tissues, the targeted areas were cut macroscopically with a fine needle referring to the microscopic observation of the morphology of serial HE sections. After the tissue sections on the glass slide were immersed in distilled water, only the targeted areas of cancer tissue were separated from the glass slide and recovered (Figure 1). Adenomas were also cut and collected in the same manner. Normal mucosa was recovered from 5-cm-long sections of full-depth colorectal wall with a fine needle. The dissected tissues were further cut into small pieces and then placed in Eppendorf tubes. Two hundred µl of RIPA buffer, pH 7.6 [1 M sodium dihydrogen phosphate, 10 mM disodium hydrogen phosphate, 154 mM sodium chloride, 1% Triton X-100, 12 mM sodium deoxycholate, 0.2% sodium azide, 0.95 mM fluoride, 2 mM phen-ylmethylsulfonyl fluoride, 50 mg/ml aprotinin (Sigma Chemical; St Louis, MO), 50 mM leupeptin (Bayer; Leverkusen, Germany)], containing 0.1% or 2% SDS, was added to each tube, and the contents were incubated under different conditions as follows: at 0C for 2 hr; at 37C for 2 hr; at 60C for 2 hr; and at 100C for 20 min, followed by incubation at 60C for 2 hr. After incubation, the tissue lysates were centrifuged at 15,000 x g for 20 min at 4C. The supernatants were collected and stored at -80C until use for protein assay and Western blot analysis.
|
From fresh cancer tissues, protein was extracted by the conventional method, in which 100 mg of the fresh frozen tissues was homogenized with a syringe-type, hand-held homogenizer and incubated in 1 ml of RIPA buffer containing 0.1% SDS at 0C. The tissue lysates from fresh frozen tissues were also centrifuged at 15,000 x g for 20 min at 4C and the supernatants were collected and stored at -80C.
Protein Assay
The protein concentration of the lysates was determined with the Bio-Rad DC Protein Assay (Bio-Rad Laboratories: Hercules, CA). The reaction is similar to the well-documented Lowry assay (
SDS-PAGE and Western Blot Analysis
The lysates were treated with SDS-PAGE loading buffer (at a final concentration of 65 mM Tris, 5% 2-mercaptoethanol, 3% SDS, and 10% glycerol) at 100C for 5 min. The total lysates extracted from a 5-mm2 cancer tissue section or a 5-cm-long normal mucosal section of 50 µm thickness were applied to each lane and electrophoresed in 12% or 7.5% polyacrylamide gel (13 x 13 cm, 1 mm thick). Fifty µg of total cellular protein of extracts from fresh frozen tissue was also applied and electrophoresed. To examine the range of molecular weight of the extracted proteins, we investigated the SDS-PAGE pattern by Coomassie blue staining of the electrophoresed gels before transfer. For Western blot analysis, the electrophoresed proteins were transferred onto an Immobilon polyvinylidene difluoride (PVDF) membrane (Millipore; Bedford, MA) in transfer buffer containing 192 mM glycine, 25 mM Tris-HCl, pH 8.3, 20% v/v methanol, and 0.02% SDS. After overnight treatment with blocking buffer (5% nonfat dry milk, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20) at 4C, the membranes were incubated with the primary antibody at room temperature (RT) for 2 hr at the following dilutions or final concentrations: anti-PCNA MAb 1:400; anti-p53 MAb 1:50; anti-cyclin D1 polyclonal antibody 0.1 µg/ml; anti-cyclin E MAb 2 µg/ml; anti-CDK2 polyclonal antibody 0.2 µg/ml; anti-CDK4 polyclonal antibody 1:500; anti-ß-catenin MAb 1:500; and anti-E-cadherin MAb 2 µg/ml. For negative control, normal mouse IgG (2 µg/ml) or normal rabbit IgG (5 µg/ml) was added instead of the primary antibodies.
After being washed with TBST (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20), the filters were incubated for 1 h with alkaline phosphatase-conjugated rabbit anti-mouse IgG or goat anti-rabbit IgG at a dilution of 1:3000 and were developed with the ProtoBlot NBT and BICP Color Development System (Promega; Madison, WI).
To test the sensitivity, sequentially diluted lysates were analyzed by Western blotting for PCNA and the minimal size of dissected tissue required was determined.
To examine the expression of cyclin D1 and CDK2 in normal mucosa, adenomas, and cancer tissues, 50 µg of total cell protein extracted from these tissue sections was applied for Western blotting of cyclin D1 and CDK2. Densitometric analysis of the Western blotting was performed with an Image Scanning device (Molecular Dynamics; Sunnyvale, CA). After the protein bands on the membrane were scanned, the density was measured. The quantity of the protein was calculated integrally. For four adenomas and cancer tissues, we compared the expression levels of cyclin D1 and CDK2 with matched normal mucosa.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Protein Extraction
The total extracted protein from 5-mm2 cancer tissue that was dissected from the same area of the serial sections was measured in six cases. This procedure was conducted under various conditions of incubation in RIPA buffer containing 0.1 or 2% SDS. Protein could not be extracted with incubation in RIPA buffer containing 0.1% SDS at 0C, 37C, or 60C for 2 hr. Preincubation at 100C for 20 min before treatment at 60C for 2 hr did not increase the efficiency of protein extraction. The next step was to extract protein from the tissue sections with incubation in RIPA buffer containing 2% SDS. For six samples, an average of only 13.6 µg of protein could be extracted at 0C, 14.3 µg at 37C, and 15.2 µg at 60C for 2-hr incubation (Table 1). On the other hand, when the sections were incubated at 100C for 20 min before incubation at 60C for 2 hr, 121.5 µg of protein was extracted from 5-mm2 50-µm-thick cancer tissue (Table 1). Because the average dry weight of 5-mm2 50-µm-thick cancer tissue was 0.74 mg for 54 samples (range 0.561.11 mg, median 0.67 mg), the efficiency of protein extraction was calculated as 164.2 µg/mg of dry cancer tissue.
|
We examined the molecular weight of extracted protein from paraffin sections compared with fresh frozen tissues. Although the SDS-PAGE pattern of the extracted protein identified with Coomassie blue staining was not identical to that obtained from fresh frozen tissue, it indicated that the protein molecules ranging from 10 kD to 120 kD remained well preserved (Figure 2A). The storage period of paraffin blocks did not affect the protein extraction or the electrophoretic pattern.
|
Western Blot Analysis
First, PCNA expression was analyzed by Western blotting of extracted proteins. As shown in Figure 2B, extracts from both normal mucosa and cancer tissue produced a strong PCNA band (36 kD). This band was comparable to that produced by the extract from fresh frozen cancer tissue. To determine the sensitivity of the method, we analyzed sequentially diluted lysates by Western blotting for PCNA. The protein band for PCNA could be detected even when the lysate extracted from 5-mm2 tissue was diluted to 1:32 (Figure 2B).
Western blotting of nuclear proteins including mutant-type p53 (53 kD), cyclin D1 (36 kD), cyclin E (52 kD), cyclin-dependent kinase 2 (CDK2, 33 kD) and cyclin-dependent kinase 4 (CDK4, 34 kD), cytosolic ß-catenin (92 kD), and membrane-bound E-cadherin (120 kD) also demonstrated clear protein bands at their expected molecular weight (Figure 3A and Figure 3B).
|
In our study, we could not detect the retinoblastoma gene product (pRB) or carcinoembryonic antigen (CEA) in the extracts from buffered formalin-fixed, paraffin-embedded tissue sections (data not shown).
Application of the New Method
We examined the expression levels of cyclin D1 and CDK2 in small adenomas and cancer tissues of the colorectum in comparison with the matched normal mucosa from four cases (Figure 4; Table 2). The definitive regions exhibiting moderate dysplasia were dissected from the adenomas in sizes ranging from 5 mm to 14 mm, from which enough protein could be extracted for Western blotting. Western blotting and densitometric analysis revealed that the average cyclin D1 expression of four adenomas was 2.1 times higher than that of matched normal mucosa, whereas CDK2 expression was only 1.4 times higher. Cyclin D1 expression in cancer tissues was 3.6 times higher and of CDK2 3.3 times higher.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Buffered formalin is a popular fixative and has been used for fixation of surgically or endoscopically resected specimens (
The efficiency of protein extraction (164.2 µg/mg of dry cancer tissue) was more efficient than that from frozen cancer tissues (44.7 µg protein/mg of frozen cancer tissue on average for 10 samples) by the conventional method of homogenization and incubation in RIPA buffer containing 0.1% SDS at 0C. Although the extraction efficiency was about 50% of that previously reported for AMeX-processed tissues (
Not only PCNA but also many kinds of nuclear, cytosolic, and membrane-bound proteins were detected at their expected molecular weight. The data indicate that the full length of these proteins was well preserved by formalin fixation and could be extracted without degradation even after several years of storage. This appears to be reasonable in view of the fact that formalin fixation does not alter the secondary protein structure, i.e., the regular arrangement of the polypeptide backbone, whereas it might change the tertiary or quaternary structure, i.e., the three-dimensional structure or the structure of aggregates of globular proteins (
To our surprise, P53, CDK4, E-cadherin, and ß-catenin, which could not be immunohistochemically detected in the formalin-fixed tissue sections by the antibodies employed in this study (
In our study we could not clearly detect the retinoblastoma gene product (pRB) or carcinoembryonic antigen (CEA) in the extracts from buffered formalin-fixed, paraffin-embedded tissue sections (data not shown). pRB is known to be present at a very low level, and it appeared necessary to apply much greater amounts of the extracts for detection of pRB. CEA is produced in large amounts in colon tissue but is tightly attached to the membrane via a glycosylphosphatidylinositol anchor (
This new technique provides many advantages for investigation of protein expression. The most valuable is the ability to extract protein from a small region under histological observation. In this study we could examine the expression levels of cyclin D1 and CDK2 in small adenomas and cancer tissues of the colorectum in comparison with the matched normal mucosa. The values for CDK2 overexpression compare well with our previous data obtained with conventional protein extraction from fresh frozen tissues (
In conclusion, the technique of protein extraction from buffered formalin-fixed, paraffin-embedded tissue described here should provide a powerful tool for protein analysis, because large-scale archives of pathological specimens routinely fixed in formalin can be made available and its application could lead to detailed analysis of protein expression.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bell PB, Rundquist I, Svensson I, Collins VP (1987) Use of cytofluorometry to evaluate binding of antibodies to the cytoskeleton of cultured cells. J Histochem Cytochem 35:1381-1388[Abstract]
Cattoretti G, Pileri S, Parravicini C, Becher MHG, Poggi S, Bifulco C, Key G, D'amato L, Sabattini E, Feudale E, Reynolds F, Gerdes J, Rilke F (1993) Antigen unmasking on formalin-fixed, paraffin-embedded tissue sections. J Pathol 171:83-98[Medline]
Cheng L, Nagabhushan M, Pretlow TP, Amini SB, Pretlow TG (1996) Expression of E-cadherin in primary and metastatic prostate cancer. Am J Pathol 148:1375-1380[Abstract]
Clark RK, Damjanov J (1986) Immunoblotting of keratin polypeptides extracted from tissues preserved in standard histologic fixatives. J Histochem Cytochem 34:679-682[Abstract]
Conti CJ, Larcher F, Chesner J, Aldaz CM (1988) Polyacrylamide gel electrophoresis and immunoblotting of proteins extracted from paraffin-embedded tissue sections. J Histochem Cytochem 36:547-550[Abstract]
Dell'Orco RT, Jupe ER, Manjeshwar WM, Lie X-T, McClung JK, King R, Hollingsworth A, Lightfoot S, Kern W (1997) Phohibitin: a new biomarker for breast tumors. Breast J 3:85-89
Fox CH, Johnson FB, Whiting J, Roller PP (1985) Formaldehyde fixation. J Histochem Cytochem 33:845-853[Medline]
Hefta SA, Hefta LJS, Lee TD, Paxton RJ, Shively JE (1988) Carcinoembryonic antigen is anchored to membranes by covalent attachment to a glycosyl-phosphatidylinositol moiety: identification of the ethanolamine linkage site. Proc Natl Acad Sci USA 85:4648-4652[Abstract]
Jean F, Malapert P, Rougon G, Barbet J (1988) Cell membrane, but not circulating carcinoembryonic antigen is linked to a phosphatidylinositol-containing hydrophobic domain. Biochem Biophys Res Commun 155:794-800[Medline]
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275
Mason JT, O'Leary TJ (1991) Effects of formaldehyde fixation on protein secondary structure: a calorimetric and infrared spectroscopic investigation. J Histochem Cytochem 39:225-229[Abstract]
Matsuoka Y, Matsuo Y, Sugano K, Ohkura H, Kuroki Mo Kuroki Ma (1990) Characterization of carcinoembryonic antigen-related antigens in normal adult feces. Jpn J Cancer Res 81:514-519[Medline]
Matsushime H, Ewen ME, Strom DK, Kato J, Hanks SK, Roussel MF, Sherr CJ (1992) Identification and properties of an atypical catalytic subunit (p34PSKJ3/CDK4) for mammalian D-type G1 cyclins. Cell 71:323-334[Medline]
Marx J (1994) How cells cycle toward cancer. Science 263:319-321[Medline]
Meyerson M, Enders GH, Wu C-L, Su L-K, Gorka C, Nelson C, Harlow E, Tsai L-H (1992) A family of human cdc2-related protein kinases. EMBO J 11:2909-2917[Abstract]
Nagafuchi A, Takeichi M (1988) Cell binding function of E-cadherin is regulated by the cytoplasmic domain. EMBO J 7:3679-3684[Abstract]
Neubauer A, Neubauer B, He M, Effert P, Iglehart D, Frye RA, Liu E (1992) Analysis of gene amplification in archival tissue by differential polymerase chain reaction. Oncogene 7:1019-1025[Medline]
Norton AJ (1993) Microwave oven heating for antigen unmasking in routinely processed tissue sections. J Pathol 171:79-80[Medline]
Ohue M, Tomita N, Monden T, Fujita M, Fukunaga M, Tamaki K, Yana I, Ohnishi T, Enomoto T, Inoue M, Shimano T, Mori T (1994) A frequent alteration of p53 gene in carcinoma in adenoma of colon. Cancer Res 54:4798-4804[Abstract]
Ozawa M, Baribault H, Keimer R (1989) The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species. EMBO J 8:1711-1717[Abstract]
Sack TL, Gum JR, Low MG, Kim YS (1988) Release of carcinoembryonic antigen from human colonic cancer cells by phosphatidylinositol-specific phospholipase C. J Clin Invest 82:586-593[Medline]
Sato Y, Mukai K, Furuya S, Kameya T, Hirohashi S (1992) The AMeX method: a multipurpose tissue-processing and paraffin-embedding method. Am J Pathol 140:775-779[Abstract]
Shi SR, Key ME, Kalra KL (1991) Antigen retrieval in formalin-fixed, paraffin-embedded tissue: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 39:741-748[Abstract]
Shimaya K, Shiozaki H, Inoue M, Tahara H, Monden T, Shimano T, Mori T (1993) Significance of p53 expression as a prognostic factor in esophageal squamous cell carcinoma. Virchows Arch [A] 422:271-276
Takami N, Misumi Y, Kuroki M, Matsuoka Y, Ikehara Y (1988) Evidence for carboxyterminal processing and glycolipid-anchoring of human carcinoembryonic antigen. J Biol Chem 263:12716-12720
Van den Berg FM, Baas IO, Polak MM, Offerhaus JA (1993) Detection of p53 overexpression in routinely paraffin-embedded tissue of human carcinomas using a novel target unmasking fluid. Am J Pathol 142:381-385[Abstract]
Vogelstein B, Fearon ER, Hamilton SR, Kern SE, Preisinger AC, Leppert M, Nakamura Y, White R, Smith AMM, Bos JL (1988) Genetic alterations during colorectal-tumor development. N Engl J Med 319:525-532[Abstract]
von Weizsäcker F, Labeit S, Koch HK, Oehlert W, Gerok W, Blum HE (1991) A simple and rapid method for the detection of RNA in formalin-fixed, paraffin-embedded tissues by PCR amplification. Biochem Biophys Res Commun 174:176-180[Medline]
Xiong Y, Connoly T, Futcher B, Beach D (1991) Human D-type cyclin. Cell 65:691-699[Medline]
Yamamoto H, Monden T, Ikeda K, Izawa H, Fukuda K, Fukunaga M, Tomita N, Shimano T, Shiozaki H, Monden M (1995) Coexpression of cdk2/cdc2 and retinoblastoma gene products in colorectal cancer. Br J Cancer 71:1231-1236[Medline]