Abstract
There are several double immunolabelling methods but each has its drawbacks. More often than not, antibodies with the required specificities are available in only one species and their use normally produces false labels due to cross-reactivity. We describe a new and reliable technique for staining with primary antibodies from the same species, that can even be employed on tissues of the donor species. The protocol avoids cross-reactivities without loss in sensitivity, uses commercially available reagents and takes advantage of enzymatic detection, although it can be adapted for fluorescent labelling. Briefly, tissue is incubated with one primary antibody, followed by a peroxidase-coupled secondary antibody which is detected using amino ethyl carbazol to give a red reaction product. Meanwhile, the next primary antibody is coupled in vitro to a biotinylated secondary antibody and excess binding sites quenched with normal immune serum from the same species as the primary antibody. This complex is applied to tissue and detected by the avidin-biotin/alkaline phosphatase technique using naphthol-AS-MX-phosphate/Fast Blue BB to produce a blue label. In addition to extensive controls, the reliability and broad applicability of this method has been confirmed in (I) murine skin cryostat sections to co-visualize antigen-presenting cells (MHC class II-immunoreactive; '-ir') with either antigen detecting T lymphocytes (CD4-ir) or Langerhans cells (NLDC-145-ir) and (2) locust (Insecta) abdominal ganglion paraffin sections, where it is known that immunoreactivities for octopamine and a FMRFamide-related peptide are colocalized in only one, uniquely identifiable neuron.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 255-265 |
| Number of pages | 11 |
| Journal | Journal of Immunological Methods |
| Volume | 190 |
| Issue number | 2 |
| DOIs | |
| State | Published - Apr 19 1996 |
| Externally published | Yes |
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Keywords
- CD4
- Colocalization
- FMRFamide
- Inscct nervous system
- Major histocompatibility complex class II
- Murine skin
- NLDC-145
- Octopamine
ASJC Scopus subject areas
- Immunology and Allergy
- Immunology
Cite this
A new method for double immunolabelling with primary antibodies from identical species. / Eichmüller, Stefan; Stevenson, Paul A.; Paus, Ralf.
In: Journal of Immunological Methods, Vol. 190, No. 2, 19.04.1996, p. 255-265.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - A new method for double immunolabelling with primary antibodies from identical species
AU - Eichmüller, Stefan
AU - Stevenson, Paul A.
AU - Paus, Ralf
PY - 1996/4/19
Y1 - 1996/4/19
N2 - There are several double immunolabelling methods but each has its drawbacks. More often than not, antibodies with the required specificities are available in only one species and their use normally produces false labels due to cross-reactivity. We describe a new and reliable technique for staining with primary antibodies from the same species, that can even be employed on tissues of the donor species. The protocol avoids cross-reactivities without loss in sensitivity, uses commercially available reagents and takes advantage of enzymatic detection, although it can be adapted for fluorescent labelling. Briefly, tissue is incubated with one primary antibody, followed by a peroxidase-coupled secondary antibody which is detected using amino ethyl carbazol to give a red reaction product. Meanwhile, the next primary antibody is coupled in vitro to a biotinylated secondary antibody and excess binding sites quenched with normal immune serum from the same species as the primary antibody. This complex is applied to tissue and detected by the avidin-biotin/alkaline phosphatase technique using naphthol-AS-MX-phosphate/Fast Blue BB to produce a blue label. In addition to extensive controls, the reliability and broad applicability of this method has been confirmed in (I) murine skin cryostat sections to co-visualize antigen-presenting cells (MHC class II-immunoreactive; '-ir') with either antigen detecting T lymphocytes (CD4-ir) or Langerhans cells (NLDC-145-ir) and (2) locust (Insecta) abdominal ganglion paraffin sections, where it is known that immunoreactivities for octopamine and a FMRFamide-related peptide are colocalized in only one, uniquely identifiable neuron.
AB - There are several double immunolabelling methods but each has its drawbacks. More often than not, antibodies with the required specificities are available in only one species and their use normally produces false labels due to cross-reactivity. We describe a new and reliable technique for staining with primary antibodies from the same species, that can even be employed on tissues of the donor species. The protocol avoids cross-reactivities without loss in sensitivity, uses commercially available reagents and takes advantage of enzymatic detection, although it can be adapted for fluorescent labelling. Briefly, tissue is incubated with one primary antibody, followed by a peroxidase-coupled secondary antibody which is detected using amino ethyl carbazol to give a red reaction product. Meanwhile, the next primary antibody is coupled in vitro to a biotinylated secondary antibody and excess binding sites quenched with normal immune serum from the same species as the primary antibody. This complex is applied to tissue and detected by the avidin-biotin/alkaline phosphatase technique using naphthol-AS-MX-phosphate/Fast Blue BB to produce a blue label. In addition to extensive controls, the reliability and broad applicability of this method has been confirmed in (I) murine skin cryostat sections to co-visualize antigen-presenting cells (MHC class II-immunoreactive; '-ir') with either antigen detecting T lymphocytes (CD4-ir) or Langerhans cells (NLDC-145-ir) and (2) locust (Insecta) abdominal ganglion paraffin sections, where it is known that immunoreactivities for octopamine and a FMRFamide-related peptide are colocalized in only one, uniquely identifiable neuron.
KW - CD4
KW - Colocalization
KW - FMRFamide
KW - Inscct nervous system
KW - Major histocompatibility complex class II
KW - Murine skin
KW - NLDC-145
KW - Octopamine
UR - http://www.scopus.com/inward/record.url?scp=18744418780&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=18744418780&partnerID=8YFLogxK
U2 - 10.1016/0022-1759(95)00281-2
DO - 10.1016/0022-1759(95)00281-2
M3 - Article
C2 - 8621960
AN - SCOPUS:18744418780
VL - 190
SP - 255
EP - 265
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
SN - 0022-1759
IS - 2
ER -