Fc-mediated nonspecific staining of the porcine brain with rabbit antisera in immunocytochemistry is prevented by pre-incubation of the sera with proteins A and G.
Nonspecific staining was detected in immunocytochemical procedures on the porcine hypothalamus with rabbit antisera, irrespective of the antigen specificity of the sera, in magnocellular neurons of the paraventricular (PVN) and supraoptic nuclei (SON), and in the vasopressin- and oxytocin-containing nucleus (VON). The present study was designed to test the hypothesis that this staining is mediated by the Fc portion of rabbit immunoglobulins. Rabbit antisera against neuropeptides localized predominantly outside the PVN, SON, and VON were employed in combination with different detection methods. The intensity of the nonspecific staining varied depending on the antiserum and persisted after pre-absorption of the antisera with their homologous peptides. Nonspecific staining and antigen-specific staining were differentially affected by the method of tissue fixation. The nonspecific staining could be prevented by preincubation of the antisera with proteins A and G, which left the antigen-specific staining intact, whereas additional preabsorption with homologous peptide abolished all staining. These observations suggest that the Fc region of IgGs is indeed involved in the nonspecific staining. On press-blots of homogenates from SON tissue subjected to isoelectric focusing, one band in the low-pH region was found with all antisera. Pre-incubation of the antisera with protein A abolished the staining of this band but did not affect staining of antigen-specific bands. Pre-incubation with proteins A and G is proposed as a routine control to check for nonspecific staining mediated by the Fc region of IgGs in immunocytochemical procedures, particularly those that employ rabbit sera in porcine brain.
van der Beek
E M
EM
Department of Cell Biology, Medical Faculty, University of Utrecht, The Netherlands.
Pool
C W
CW
van Eerdenburg
F J
FJ
Sluiter
A A
AA
van der Donk
H A
HA
van den Hurk
R
R
Wiegant
V M
VM
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism*; D005260:Female; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism*; D007150:Immunohistochemistry; D007525:Isoelectric Focusing; D011817:Rabbits / Q000276:immunology; D013194:Staining and Labeling / Q000379:methods*; D013205:Staphylococcal Protein A / Q000378:metabolism*; D013291:Streptococcus / Q000378:metabolism*; D013552:Swine
D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D007141:Immunoglobulin Fc Fragments; D013205:Staphylococcal Protein A