Ultrastructural and nuclear antigen preservation after high-pressure freezing/freeze-substitution and low-temperature LR White embedding of HeLa cells.
A protocol for high-pressure freezing and LR White embedding of mammalian cells suitable for fine ultrastructural studies in combination with immunogold labelling is presented. HeLa S3 cells enclosed in low-temperature gelling agarose were high-pressure frozen, freeze-substituted in acetone, and embedded in LR White at 0 degrees C. The morphology of such cells and the preservation of nuclear antigens were excellent in comparison with chemically fixed cells embedded in the same resin. The immunolabelling signal for different nuclear antigens was 4-to-13 times higher in high-pressure frozen than in chemically fixed cells. We conclude that one can successfully use high-pressure freezing/freeze-substitution and LR White embedding as an alternative of Lowicryl resins.
Strádalová
Vendula
V
Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, v.v.i., Vídenská 1083, 142 20, Prague, Czech Republic.
Gaplovská-Kyselá
Katarína
K
Hozák
Pavel
P
D000180:Acrylic Resins; D034961:Antigens, Nuclear / Q000032:analysis*; D002467:Cell Nucleus / Q000276:immunology* / Q000648:ultrastructure*; D015925:Cryopreservation / Q000379:methods*; D017110:Freeze Substitution; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D046529:Microscopy, Electron, Transmission; D016613:Plastic Embedding / Q000379:methods*; D011312:Pressure
D000180:Acrylic Resins; D034961:Antigens, Nuclear; C048707:LR white
Methods Enzymol. 2002;351:96-123
12073378
J Microsc. 2003 Oct;212(Pt 1):13-20
14516357
J Microsc. 2003 Oct;212(Pt 1):44-52
14516361
J Microsc. 2003 Oct;212(Pt 1):53-61
14516362
Glycobiology. 1996 Dec;6(8):817-22
9023543
J Microsc. 1995 Sep;179(Pt 3):321-32
7473694
Histochemistry. 1992;97(5):421-9
1500298
Anat Rec A Discov Mol Cell Evol Biol. 2006 Oct;288(10):1059-67
16952173
Eur J Histochem. 2002;46(4):359-64
12597621
Histochem Cell Biol. 1997 Apr;107(4):303-12
9151112
J Microsc. 2002 Oct;208(Pt 1):3-10
12366592
J Microsc. 1990 Jun;158(Pt 3):355-63
1697624
J Microsc. 2004 Jan;213(1):81-5
14678516
J Microsc. 1994 Jul;175(Pt 1):34-43
7932676
J Microsc. 2002 Dec;208(Pt 3):158-66
12460447
J Electron Microsc Tech. 1989 Nov;13(3):165-74
2685196
J Histochem Cytochem. 2004 Jan;52(1):77-86
14688219
Exp Dermatol. 2004 Jul;13(7):419-25
15217362
Microsc Res Tech. 2001 May 15;53(4):313-21
11340677
Methods Cell Biol. 1989;31:513-51
2674631
Scanning Microsc Suppl. 1989;3:253-68; discussion 268-9
2694271
Methods Mol Biol. 1999;117:77-97
10327400
Biol Cell. 1996;86(1):1-10
8688826
Microsc Res Tech. 1995 Aug 15;31(6):531-2
8527853
Micron. 2001 Jan;32(1):11-9
10900376
Microsc Res Tech. 1993 Apr 15;24(6):474-87
8490233
Biol Cell. 1991;72(1-2):113-9
1756301
Eur J Histochem. 1993;37(1):5-18
7682867
J Microsc. 2003 Oct;212(Pt 1):21-5
14516358
J Microsc. 1998 Dec;192(Pt 3):248-58
9923417