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Immunohistochemistry of mucin core proteins
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Immunohistochemistry of mucin core proteins

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Category
Sugar binding proteins
Protocol Name

Immunohistochemistry of mucin core proteins

Authors
 Tian, Yuan
Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, The University of Tokyo

Irimura, Tatsuro *
Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, The University of Tokyo
*To whom correspondence should be addressed.
KeyWords
Reagents
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Preparation of polyclonal antisera specific for the cytoplasmic tail of human MUC21

  1. A synthetic oligopeptide (C)RPVSSIAMEMSGRNSGP corresponding to the 17 residues at the carboxyl terminal of MUC21 is prepared by the use of peptide synthesizer (Applied Biosystems/Life Technologies, Carlsbad, CA) and purified by high performance liquid chromatography equipped with a C18 reverse-phase column (Nacalai Tesque Inc., Kyoto, Japan).
  2. The size of the oligopeptide is assessed by MALDI-TOF mass-spectroscopy with Voyager Elite.
  3. The peptide (1 mg) is conjugated to keyhole limpet hemocyanin (20 mg: Sigma-Aldrich, St. Louis, MO) with m-maleimidobenzoyl-N-hydroxysuccinimide ester through the attached cysteine residue added at the amino terminus.
  4. Japanese white rabbits are immunized twice with the conjugate with a two-week interval.
  5. The sera are obtained when the antibody titer is 2.4 × 104 (to reach a half maximum binding to the peptide) in ELISA two weeks after the second immunization. This antibody is named as anti-MUC21CT (Itoh Y. et al. 2008).
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Preparation of antibodies specific for human MUC1

  1. Preparations of antibodies specific for MUC1 are described elsewhere (Yamamoto M. et al. 1996; Higuchi T. et al. 2000)
Methods
1.

Immunohistological staining of malignant and non-malignant tissue sections by the use of polyclonal antiserum specific for the cytoplasmic tail of MUC21

1) 

 Tissues are preserved in 10% buffered formalin and embedded in paraffin and are serially sectioned at 4 μm thickness, mounted on silane-coated slides, and deparaffinized.

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2) 

 The slides with sections are immersed for 30 min in 1% hydrogen peroxide in PBS to inactivate endogenous peroxidase.

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3) 

 After washing 3 times with PBS for 5 min, the sections are incubated with 2% normal goat serum-3% bovine serum albumin/PBS (2% NGS-3% BSA/PBS) containing avidin (Vector Laboratories, Inc., Burlingame, CA) to block nonspecific antibody binding for 30 min at room temperature in a humidified chamber, then reacted with rabbit polyclonal antibody specific for cytoplasmic tail of human MUC21 (anti-MUC21CT: 1.4 mg/mL) at 1:1000 dilution in PBS containing 2% NGS-3% BSA with biotin (Vector Laboratories) overnight at 4°C.

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4) 

 After washing with PBS, biotin labeled goat anti-rabbit IgG (0.75 mg/mL) was added at 1: 200 dilution in 2% NGS-3% BSA/PBS as the second antibody.

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5) 

 The antibody binding was visualized by the use of peroxidase-conjugated streptavidin (1.25 mg/mL) at 1: 100 dilution in 2% NGS-3% BSA/PBS following incubation with diethylaminobenzidine (Vector Laboratories).

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2.

Immunohistological staining of tissue sections by the use of antibodies specific for different domains of MUC1

1) 

 Two antibodies, mAb MY.1E12 (anti-MUC1 with sialyl-T antigen) (Yamamoto M. et al. 1996; Takeuchi H. et al. 2002) and hen polyclonal IgY antibody specific for synthetic peptide corresponding to the cytoplasmic portion (carboxyl terminal) of MUC1, SSLSYTNPAVAATSANL (Higuchi T. et al. 2000), are used.

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2) 

 OCT-embedded frozen tissues from normal pancreas of a MUC1-transgenic mouse are serially sectioned at 5 μm, mounted on poly-L-lysine-coated slides, and fixed in cold ethanol for 30 sec.

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3) 

 Slides are washed 3 times for 5 min each in PBS, and immersed in 0.3% hydrogen peroxide in PBS 3 times for 5 min to inactivate endogenous peroxidase.

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4) 

 The sections are incubated with avidin diluted in blocking solution (2% NGS, 2% normal mouse serum [NMS], 3% BSA in PBS) at room temperature for 15 min, washed 3 times for 5 min each in PBS, then incubated with biotin in the blocking solution for 15 min, and washed 3 times for 5 min each in PBS.

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5) 

 The sections are incubated with 5 μg/mL of biotin conjugated mAb MY.1E12, or biotin conjugated hen polyclonal IgY antibody as primary antibodies at 4°C overnight.

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6) 

 Biotin conjugated-IgG2a (BioLegend, San Diego, CA) (5 μg/mL) or biotin-conjugated IgY (Santa Cruz Biotechnology, Santa Cruz, CA) (5 μg/mL) are used as isotype controls.

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7) 

 The sections are washed with PBS, incubated with HRP-avidin (Vector Laboratories) diluted by of 1:200 in blocking solution at room temperature for 30 min, washed, incubated with diaminobenzidine staining kit (Vector Laboratories) until a sufficient color developed, counterstained in Mayer’s Hematoxylin, and mounted in Entellan Neu (Merck Millipore, Billerica, MA).

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Figure & Legends

Figure & Legends

 

Mucin Antibody Epitope Source / Reference
 MUC2      3A2  VNTR domain  Jass JR, University of Queensland,  Australia. (winterford et al., 1999)
 Ccp58  VNTR domain  Novocastra  UK (Glickman et al., 2003)
 PH1417  C-terminus (aa 4995–5013)  G. Hansson, Go¨teburg University,  Sweden  (Axelsson et al., 1998)
 PH1491  N-terminus (aa 1167–1180)  G. Hansson, Go¨teburg University,  Sweden  (Asker et al., 1998)
 H-300 (polyclonal)  amino acids 4880-5179  mapping at the C-terminus  Santa Cruz Biotechnology, USA
 MUC3     M3.1 (ab24068)  TR  Abcam, UK
 N-20 (polyclonal)  peptide mapping within an  internal region of Mucin 3A  Santa Cruz Biotechnology, USA
 SPM200  TR  Santa Cruz Biotechnology, USA
 3H2744  TR  Santa Cruz Biotechnology, USA
 MUC4    8G7  TR  Batra SK, University of Nebraska Medical  Center, Nebraska (Moniaux et al., 2004)
 M4.275  VNTR domain  Jass JR, University of Queensland,  Australia. (winterford et al., 1999)
 1G8  Ectodomain of ASGP-2  Zymed, San Francisco, USA (Zhang et al.,  2005)
 MUC5AC     CLH2  TR  U. Mandel, University of Copenhagen,  Denmark (Reis et al., 1997); Vector Labs,  Burlingame, CA
 45M1  core polypeptide  Novocastra  UK (Glickman et al., 2003)
 1-13M1  N-terminus: cys-2, cys-4  domains  NeoMarkers, Fremont, USA (Nollet et al.,  2004)
 791  D3 domin  I.K. Gipson (Argueso et al., 2002)
 MUC5B     799  D4 domain  I.K. Gipson (Gipson et al., 2001)
 Eu-MUC5Ba  Cysteine-rich domain  D.M. Swallow, University College, London,  UK (Rousseau et al., 2003)
 H-300 (polyclonal)  amino acids 1201-1500  mapping near the C-terminus  Santa Cruz Biotechnology, USA
 4H310

 a peptide mapping at the

 N-terminus

  Santa Cruz Biotechnology, USA
 MUC6   CLH5  core polypeptide  Novocastra,  UK (Glickman et al., 2003)
 0.N.459  TR  Santa Cruz biotechnology, USA
 MUC7    PANH3  N-terminus (aa 72-92)  U. Mandel, University of Copenhagen,  Denmark (Nielsen et al., 1996)
 Eu-MUC7a  Histatin-like domain  D.M. Swallow, University College, London,  UK (Rousseau et al., 2003)
 H-150( polyclonal)  amino acids 1-75 mapping at  the N-terminus  Santa Cruz Biotechnology, USA
 MUC12   ab2298 (polyclonal)  amino acids 541-555  Abcam UK (Williams et al., 1999)
 ab3162 (polyclonal)  SSISGEPTSLYSQAE  Abcam UK (Williams et al., 1999)
 MUC13       ppz0020  EGF2 domain

 Aburatani H, University of Tokyo, Japan

 (Shimamura et al., 2005)
 ppz0025  N-terminus

 Aburatani H, University of Tokyo, Japan

 (Shimamura et al., 2005)
 ST0751(polyclonal)  C-terminus  Aburatani H, University of Tokyo, Japan (Shimamura et al., 2005)
 ab65109 (polyclonal)  internal sequence  Abcam, UK
 ab72050 (polyclonal)  residues 480-530  Abcam, UK
 H-300 (polyclonal)

 amino acids 19-318 mapping  within an N-terminal  extracellular domain

  Santa Cruz Biotechnology, USA
 MUC15    HPA026110  (polyclonal)                                 Tag (PrEST) antigen sequence  Sigma-Aldrich
 C-15(polyclonal)  C-terminal cytoplasmic domain  Santa Cruz Biotechnology, USA
 H-210 (polyclonal)  amino acids 24-233 mapping  within an N-terminal  extracellular domain  Santa Cruz Biotechnology, USA
 MUC16    OC125  TR  Dako Corporation, Carpenteria, USA (Bast  et al., 1981)
 M11  TR  NeoMarkers, Fremont, USA (Nustad et al.,  1996)
 H-150 (polyclonal)  amino acids 6419-6568  mapping near the C-terminus  Santa Cruz Biotechnology, USA
 MUC18     ab28360 (polyclonal)  amino acids 433-647  Abcam, UK (Frittsche et al., 2008)
 ab75862(polyclonal)  amino acid residues  surrounding Y641 of human  CD146  Abcam, UK
 EPR3208(ab75769)  intracellular sequence  Abcam, UK
 N1238(ab49492)  external domain  Abcam, UK
 MUC20   ab73043 (polyclonal)  Full length protein,  corresponding to amino acids  1-538  Abcam, UK
 13380-1-AP (polyclonal)  255-555aa  Proteintech Group, USA

 

Table 1  List of antibodies specific for core polypeptides of human mucins 

 

 

Fig. 1. Immunohistochemical staining of human tissue with anti-MUC21CT

(A) Papillary carcinoma of thyroid showing strong antibody reactivity to carcinoma cells. The site of antibody binding appeared to be the cytoplasm.  (B) The antibody did not bind normal thyroid follicles adjacent to the papillary carcinoma.  (C) A section of papillary carcinoma of thyroid representing the same area as (A) was reacted with rabbit IgG.  (D) A section of normal thyroid follicles reacted with rabbit IgG. The bar indicates 100 μm. We thank Dr. Toshihisa Ogawa of Department of Breast and Endocrine Surgery, Faculty of Medicine, the University of Tokyo for providing the thyroid sections.

 

Fig. 2. Immunohistochemical staining of normal pancreas of MUC1 transgenic mouse with anti-MUC1 antibodies

(A) A section of pancreatic duct stained with purified polyclonal hen antibody specific for carboxyl terminal of human MUC1.  (B) A section of pancreatic duct was reacted with mAb MY.1E12 specific for MUC1 with sialyl-T epitope.  The bar indicates 100 μm.  Binding sites of antibody specific for C-terminal portion of human MUC1exhibied cytoplasmic distribution whereas binding sites of the antibody specific for human MUC1 with sialyl-T epitope distributed on the luminal surfaces.

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