Abstract
We applied the heat-induced antigen retrieval (HIAR) to aldehyde-fixed fresh frozen sections based on a new approach (i.e., a rapid and complete immobilization of antigen followed by heating). Frozen sections were fixed with 10% formalin in 0.1 M cacodylate buffer (pH 7.4) containing 25 mM CaCl 2 for 30 min, or with 0.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 min at room temperature, and then autoclaved in 20 mM Tris-HCl buffer (pH 9.0) for 10 min at 120C. Both fixatives yielded good tissue structure after autoclaving. In the sections fixed with formalin containing CaCl 2, 20 of 22 antigens located in the nucleus, cytoplasm, membranes, and extracellular matrix greatly recovered their antigenicity after autoclaving; only two antigens exhibited stronger immunoreaction in acetone-fixed fresh frozen sections than these sections. Heating also retrieved the immunoreactivity of at least 14 antigens in the sections fixed with glutaraldehyde. We used the similar procedures to localize ligand-free estrogen receptor α (ERα) and glucocorticoid receptors (GR). Mouse uterine cells exhibited almost the same nuclear ERα immunostaining regardless of the hormonal status in glutaraldehyde-fixed fresh frozen sections and unliganded GR was localized mainly in the nucleus of mouse hepatocytes in fresh frozen sections fixed with 20% formalin containing 50 or 75 mM CaCl2 at 40C, after autoclaving. These results demonstrate that HIAR is useful for the immunohistochemistry of many antigens in aldehyde-fixed fresh frozen sections.
| Original language | English |
|---|---|
| Pages (from-to) | 1421-1432 |
| Number of pages | 12 |
| Journal | Journal of Histochemistry and Cytochemistry |
| Volume | 53 |
| Issue number | 11 |
| DOIs | |
| Publication status | Published - 2005 Nov |
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Keywords
- Fresh frozen section
- Heat-induced antigen retrieval
- Immunohistochemistry
- Unoccupied steroid hormone receptors
ASJC Scopus subject areas
- Anatomy
- Cell Biology
Cite this
Application of heat-induced antigen retrieval to aldehyde-fixed fresh frozen sections. / Yamashita, Shuji; Okada, Yasunori.
In: Journal of Histochemistry and Cytochemistry, Vol. 53, No. 11, 11.2005, p. 1421-1432.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Application of heat-induced antigen retrieval to aldehyde-fixed fresh frozen sections
AU - Yamashita, Shuji
AU - Okada, Yasunori
PY - 2005/11
Y1 - 2005/11
N2 - We applied the heat-induced antigen retrieval (HIAR) to aldehyde-fixed fresh frozen sections based on a new approach (i.e., a rapid and complete immobilization of antigen followed by heating). Frozen sections were fixed with 10% formalin in 0.1 M cacodylate buffer (pH 7.4) containing 25 mM CaCl 2 for 30 min, or with 0.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 min at room temperature, and then autoclaved in 20 mM Tris-HCl buffer (pH 9.0) for 10 min at 120C. Both fixatives yielded good tissue structure after autoclaving. In the sections fixed with formalin containing CaCl 2, 20 of 22 antigens located in the nucleus, cytoplasm, membranes, and extracellular matrix greatly recovered their antigenicity after autoclaving; only two antigens exhibited stronger immunoreaction in acetone-fixed fresh frozen sections than these sections. Heating also retrieved the immunoreactivity of at least 14 antigens in the sections fixed with glutaraldehyde. We used the similar procedures to localize ligand-free estrogen receptor α (ERα) and glucocorticoid receptors (GR). Mouse uterine cells exhibited almost the same nuclear ERα immunostaining regardless of the hormonal status in glutaraldehyde-fixed fresh frozen sections and unliganded GR was localized mainly in the nucleus of mouse hepatocytes in fresh frozen sections fixed with 20% formalin containing 50 or 75 mM CaCl2 at 40C, after autoclaving. These results demonstrate that HIAR is useful for the immunohistochemistry of many antigens in aldehyde-fixed fresh frozen sections.
AB - We applied the heat-induced antigen retrieval (HIAR) to aldehyde-fixed fresh frozen sections based on a new approach (i.e., a rapid and complete immobilization of antigen followed by heating). Frozen sections were fixed with 10% formalin in 0.1 M cacodylate buffer (pH 7.4) containing 25 mM CaCl 2 for 30 min, or with 0.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 min at room temperature, and then autoclaved in 20 mM Tris-HCl buffer (pH 9.0) for 10 min at 120C. Both fixatives yielded good tissue structure after autoclaving. In the sections fixed with formalin containing CaCl 2, 20 of 22 antigens located in the nucleus, cytoplasm, membranes, and extracellular matrix greatly recovered their antigenicity after autoclaving; only two antigens exhibited stronger immunoreaction in acetone-fixed fresh frozen sections than these sections. Heating also retrieved the immunoreactivity of at least 14 antigens in the sections fixed with glutaraldehyde. We used the similar procedures to localize ligand-free estrogen receptor α (ERα) and glucocorticoid receptors (GR). Mouse uterine cells exhibited almost the same nuclear ERα immunostaining regardless of the hormonal status in glutaraldehyde-fixed fresh frozen sections and unliganded GR was localized mainly in the nucleus of mouse hepatocytes in fresh frozen sections fixed with 20% formalin containing 50 or 75 mM CaCl2 at 40C, after autoclaving. These results demonstrate that HIAR is useful for the immunohistochemistry of many antigens in aldehyde-fixed fresh frozen sections.
KW - Fresh frozen section
KW - Heat-induced antigen retrieval
KW - Immunohistochemistry
KW - Unoccupied steroid hormone receptors
UR - http://www.scopus.com/inward/record.url?scp=27244433299&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=27244433299&partnerID=8YFLogxK
U2 - 10.1369/jhc.4A6579.2005
DO - 10.1369/jhc.4A6579.2005
M3 - Article
C2 - 16046672
AN - SCOPUS:27244433299
VL - 53
SP - 1421
EP - 1432
JO - Journal of Histochemistry and Cytochemistry
JF - Journal of Histochemistry and Cytochemistry
SN - 0022-1554
IS - 11
ER -