scholarly journals A simple method for immunofluorescent double staining with primary antisera from the same species.

1993 ◽  
Vol 41 (4) ◽  
pp. 627-630 ◽  
Author(s):  
S Würden ◽  
U Homberg
Keyword(s):  
Schistocerca Gregaria ◽  
Staining Procedure ◽  
Double Staining ◽  
Double Immunofluorescence ◽  
Double Labeling ◽  
Simple Method ◽  
Fluorescent Markers ◽  
Secondary Antiserum ◽  
Secondary Antibodies ◽  
Texas Red

We have developed a new double immunofluorescence technique by which two neuroactive substances in the same tissue section can be labeled with primary antisera raised in the same species. The optic lobes of the locust Schistocerca gregaria were used as a model system to develop the staining procedure. FMRFamide-immunoreactive neurons were detected by rabbit antisera against FMRFamide and FITC-conjugated secondary antibodies. Antibodies against the second peptide, pigment-dispersing hormone (PDH), also raised in rabbit, were biotinylated and detected via streptavidin-Texas Red. Crossreactivity of the PDH immunoglobulins with the FITC-conjugated secondary antiserum was prevented by pre-incubation with rabbit gamma globulins. The two peptide immunoreactivities could be conveniently observed on the same section with the different fluorescent markers. This double labeling technique with modified antibodies is easily performed and highly useful for co-localization studies with antisera raised in the same species.

A pre-embedding double staining procedure used to observe the effect of fixation on the activity of intercellular adhesion molecule on T and B cells

1990 ◽  
Vol 48 (3) ◽  
pp. 378-379
Author(s):  
Jane E. Ramberg ◽  
Shigeto Tohma ◽  
Peter E. Lipsky
Keyword(s):  
B Cells ◽  
Adhesion Molecule ◽  
Colloidal Gold ◽  
Intercellular Adhesion ◽  
Intercellular Adhesion Molecule ◽  
Staining Procedure ◽  
Double Staining ◽  
T And B Cells ◽  
Microtiter Plates ◽  
Streptavidin Peroxidase

Intercellular adhesion molecule (ICAM-1) appears to be a ligand for LFA-1 dependent adhesion in T cell mediated cytotoxcity. It is found on cells of both hematopoietic and non-hematopoietic origin. While observing the activity of ICAM-1 on the surfaces of interacting T and B cells, we found that we could successfully carry out a pre-embedding double staining procedure utilizing both colloidal gold and peroxidase conjugated reagents.On 24-well microtiter plates, mitomycin-treated T4 cells were stimulated with 64.1 (anti-CD3) for one hour before the addition, in some instances, of B cells. Following a 12-48 hour incubation at 38°C, the cells were washed and then immunostained with a colloidal gold conjugated RFB-4 (anti-CD22); biotinylated R6.5 (anti-ICAM-1); followed by streptavidin/peroxidase. This method allowed us to observe two different antigens without concern about possible cross-reaction of reagents. Because we suspected ICAM-1 and R6.5 were sensitive to fixation, we tried varying concentrations of fresh paraformaldehyde before R6.5, after R6.5 and after streptavidin/peroxidase. All immunostaining and washing was done on ice with ice cold reagents.

scholarly journals Double-staining procedure for the fluorescent treponemal antibody absorption (FTA-ABS) test.

1979 ◽  
Vol 55 (2) ◽  
pp. 105-108
Author(s):  
E F Hunter ◽  
R M McKinney ◽  
S E Maddison ◽  
D D Cruce
Keyword(s):  
Staining Procedure ◽  
Double Staining ◽  
Antibody Absorption

Immunochemical Detection and Isolation of DNA from Metabolically Active Bacteria

1999 ◽  
Vol 65 (3) ◽  
pp. 1207-1213 ◽  
Author(s):  
Ena Urbach ◽  
Kevin L. Vergin ◽  
Stephen J. Giovannoni
Keyword(s):  
Monoclonal Antibodies ◽  
Microbial Community ◽  
Natural Populations ◽  
Brdu Incorporation ◽  
Pcr Analysis ◽  
Microbial Cells ◽  
Paramagnetic Beads ◽  
Specific Species ◽  
Secondary Antibodies ◽  
Texas Red

ABSTRACT Most techniques used to assay the growth of microbes in natural communities provide no information on the relationship between microbial productivity and community structure. To identify actively growing bacteria, we adapted a technique from immunocytochemistry to detect and selectively isolate DNA from bacteria incorporating bromodeoxyuridine (BrdU), a thymidine analog. In addition, we developed an immunocytochemical protocol to visualize BrdU-labeled microbial cells. Cultured bacteria and natural populations of aquatic bacterioplankton were pulse-labeled with exogenously supplied BrdU. Incorporation of BrdU into microbial DNA was demonstrated in DNA dot blots probed with anti-BrdU monoclonal antibodies and either peroxidase- or Texas red-conjugated secondary antibodies. BrdU-containing DNA was physically separated from unlabeled DNA by using antibody-coated paramagnetic beads, and the identities of bacteria contributing to both purified, BrdU-containing fractions and unfractionated, starting-material DNAs were determined by length heterogeneity PCR (LH-PCR) analysis. BrdU-containing DNA purified from a mixture of DNAs from labeled and unlabeled cultures showed >90-fold enrichment for the labeled bacterial taxon. The LH-PCR profile for BrdU-containing DNA from a labeled, natural microbial community differed from the profile for the community as a whole, demonstrating that BrdU was incorporated by a taxonomic subset of the community. Immunocytochemical detection of cells with BrdU-labeled DNA was accomplished by in situ probing with anti-BrdU monoclonal antibodies and Texas red-labeled secondary antibodies. Using this suite of techniques, microbial cells incorporating BrdU into their newly synthesized DNA can be quantified and the identities of these actively growing cells can be compared to the composition of the microbial community as a whole. Since not all strains tested could incorporate BrdU, these methods may be most useful when used to gain an understanding of the activities of specific species in the context of their microbial community.

Immunocytochemical double staining with the ELF method

1995 ◽  
Vol 53 ◽  
pp. 822-823
Author(s):  
D. G. Baskin ◽  
U C. Vathanaprida ◽  
W. L. Stahl
Keyword(s):  
Room Temperature ◽  
Immunocytochemical Staining ◽  
Double Staining ◽  
Green Fluorescence ◽  
Paraffin Sections ◽  
Double Labeling ◽  
Rat Pancreas ◽  
Bright Yellow ◽  
Green Label ◽  
Phosphatase Substrate

The reagent 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-[3H]-quinazolinone has recently become commercially available as the ELF (enzyme-labeled-fluorescence) phosphatase substrate. The ELF method is basically an alkaline phosphatase enzyme immunostaining procedure in which the reaction product is fluorescent instead of chromogenic. The ELF substrate produces a photostable, bright yellow-green fluorescence (500-580 nm) with low background. The use of the ELF substrate as a green label may have advantages over FITC in double-labeling immunocytochemical staining procedures that employ Cy3, which frequently shows residual orange-red fluorescence when viewed with conventional FITC filter sets.To evaluate the suitability of ELF for dual immunofluorescence with Cy3-labeled second antisera, we used paraffin sections of rat pancreas fixed in 4% paraformaldehyde. Primary antibodies were an anti-insulin monoclonal antibody (1:50) (Biogenex) and anti-glucagon polyclonal antibody (1:1K)2, in Tris-HCL, 1% BSA, 0.1% TX-100, pH 7.4. The insulin antibody was applied overnight at 6 °C, followed by goat anti-mouse IgGCy3 (Jackson Immunochemical Research) for 60 min. at room temperature (RT).

Immunolocalization of cytoplasmic dynein to lysosomes in cultured cells

1992 ◽  
Vol 101 (1) ◽  
pp. 125-137 ◽  
Author(s):  
S.X. Lin ◽  
C.A. Collins
Keyword(s):  
Cultured Cells ◽  
Fibroblast Cell ◽  
Cytoplasmic Dynein ◽  
Rat Tissues ◽  
Mitotic Apparatus ◽  
Specific Staining ◽  
Directed Movement ◽  
Double Labeling ◽  
Fluorescent Markers ◽  
High Degree

Polyclonal antisera have been raised against cytoplasmic dynein purified from calf brain and rat testis. These antibodies reacted most strongly with the 74 kDa dynein intermediate chain, but also recognized the 410 kDa heavy chain, and the 150 and 45 kDa polypeptides previously observed to copurify with cytoplasmic dynein from rat tissues. Localization studies were performed by indirect immunofluorescence microscopy using a fibroblast cell line. Dynein-specific staining appeared vesicular, distributed throughout the cell, but more concentrated near the nucleus. Double-labeling studies using fluorescent markers for membranous organelles indicated a co-localization of dynein with lysosomes. The distribution of the dynein-positive lysosomes was disrupted by treatment of the cells with microtubule-active drugs, and by acidification of the cytoplasm. Comparison of the distribution of lysosomes with peripheral microtubules indicated a high degree of coincidence. These results are consistent with the hypothesis that cytoplasmic dynein is involved in retrograde-directed movement of membranous organelles. In mitotic cells, dynein staining was also apparent along the microtubules of the mitotic apparatus, though vesicular staining was still conspicuous. The presence of dynein on vesicles as well as on spindle microtubules indicates that dynein distribution between these compartments may be regulated by distinct binding proteins.

scholarly journals An indirect immunofluorescence double staining procedure for the simultaneous flow cytometric measurement of iodo- and chlorodeoxyuridine incorporated into DNA

Cytometry ◽  
1991 ◽  
Vol 12 (4) ◽  
pp. 366-372 ◽  
Author(s):  
P. J. M. Bakker ◽  
J. Stap ◽  
C. J. Tukker ◽  
C. H. van Oven ◽  
C. H. N. Veenhof ◽  
...  
Keyword(s):  
Indirect Immunofluorescence ◽  
Staining Procedure ◽  
Double Staining ◽  
Flow Cytometric ◽  
Flow Cytometric Measurement

scholarly journals KIT receptor-positive cells in the bovine corpus luteum are primarily theca-derived small luteal cells

Reproduction ◽  
2007 ◽  
Vol 134 (4) ◽  
pp. 625-634 ◽  
Author(s):  
Katharina Spanel-Borowski ◽  
Kristina Sass ◽  
Sabine Löffler ◽  
Elke Brylla ◽  
Michiharu Sakurai ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Corpus Luteum ◽  
Double Staining ◽  
Double Immunofluorescence ◽  
Specific Enzyme ◽  
Thecal Cell ◽  
Luteal Cells ◽  
Bovine Corpus Luteum ◽  
Kit Receptor ◽  
Capillary Bed

The tyrosine kinase KIT receptor, the protooncogene CD117, plays a key role in growth and maturation of oocytes and follicles. Relevant data are sparse for the corpus luteum (CL). We first confirmed the presence ofKITmRNA and KIT protein in bovine CL homogenates. We then localized KIT-positive (KIT+) cells in CL sections by immunohistochemistry. At the CL stage of early development, the former theca transforming into capsule/septa showed a strong band-like KIT+ immunoresponse. In addition, CD45+ leukocytes in septa included subpopulations of CD45+/KIT+ and CD14+/KIT+ leukocytes as validated by double immunofluorescence localization. At the early secretory stage, KIT+ cells appeared within the septa/capsule region and in the periphery of the CL parenchyma, there forming a complex network. This was separate from the capillary bed as determined by double staining for CD117 and FVIII-related endothelial cell antigen (FVIIIr). The KIT+ network coincided with cells positive for cytochrome P450 17α-hydroxylase, a thecal cell-specific enzyme. The late secretory stage was defined by an advanced manifestation of the KIT+ network in the CL periphery. At the stage of regression, the KIT+ network was absent. The CL of pregnancy expressed high levels ofKITmRNA and KIT protein uniformly throughout pregnancy. The KIT+ immunolocalization revealed small fibroblast-like cells, luteal cells with granules, and clusters of large luteal cells with staining of the cell membrane. We conclude that a majority of KIT+ cells in the bovine CL are primarily theca-derived small luteal cells, and that a minority represent KIT+ leukocytes, in some cases KIT+ monocytes.

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