Indirect Immunofluorescence of Tissue Sections

2021 ◽  
pp. 17-26
Author(s):  
Cody J. Aros
Keyword(s):  
Indirect Immunofluorescence ◽  
Tissue Sections

Multiple Iteractive Labeling by Antibody Neodeposition (MILAN)

2019 ◽  
Author(s):  
Giorgio Cattoretti ◽  
Francesca Maria Bosisio ◽  
Lukas Marcelis ◽  
Maddalena Maria Bolognesi
Keyword(s):  
Image Analysis ◽  
Tissue Section ◽  
Indirect Immunofluorescence ◽  
Analysis Software ◽  
Tissue Sections ◽  
Image Analysis Software ◽  
Single Section ◽  
Fluorescent Image ◽  
Secondary Antibodies

Abstract Multiplexing, labeling for multiple immunostains the very same cell or tissue section in situ, is of considerable interest. The major obstacles to the diffusion of this technique are high costs in custom antibodies and instruments, low throughput, scarcity of specialized skills or facilities. We have validated and detail here a method based on common primary and secondary antibodies, diffusely available fluorescent image scanners and routinely processed tissue sections \(FFPE). It entails rounds of four-color indirect immunofluorescence, image acquisition and removal \(stripping) of the antibodies, before another stain is applied. The images are digitally registered and the autofluorescence is subtracted. Removal of antibodies is accomplished by disulphide cleavage. In excess of 50 different antibody stains can be applied to one single section from routinely fixed and embedded tissue. This method requires a modest investment in hardware and materials and uses freeware image analysis software.

Tissue distribution of sulfolipids in the rat. Restricted location of sulfatoxygalactosylacylalkylglyerol

1981 ◽  
Vol 59 (7) ◽  
pp. 556-563 ◽  
Author(s):  
Clifford Lingwood ◽  
Genevieve Hay ◽  
Harry Schachter
Keyword(s):  
Tissue Distribution ◽  
Indirect Immunofluorescence ◽  
Rat Tissues ◽  
Tissue Sections ◽  
Frozen Tissue

Eleven rat tissues (excluding brain) have been assayed for their ability to synthesize sulfatoxygalactosylacylalkylglycerol (SGG) from Na235SO4in vivo. These tissues were also assayed for the presence of SGG by an indirect immunofluorescence procedure using rabbit anti-SGG and frozen tissue sections. By both procedures SGG was found to be restricted to the testis; several novel sulfolipids were detected during this study.

scholarly journals Immunolocalization of Gla proteins (osteocalcin) in rat tooth germs: comparison between indirect immunofluorescence, peroxidase-antiperoxidase, avidin-biotin-peroxidase complex, and avidin-biotin-gold complex with silver enhancement.

1987 ◽  
Vol 35 (8) ◽  
pp. 825-830 ◽  
Author(s):  
A L Bronckers ◽  
S Gay ◽  
R D Finkelman ◽  
W T Butler
Keyword(s):  
Blood Vessels ◽  
Indirect Immunofluorescence ◽  
Staining Intensity ◽  
Gold Complex ◽  
Silver Enhancement ◽  
Tissue Sections ◽  
Weak Staining ◽  
Mode Of Transport ◽  
Tooth Germs ◽  
Odontoblast Processes

Odontoblasts and osteoblasts synthesize gamma-carboxyglutamatic acid (Gla)-containing proteins which are partially deposited in the mineralizing tissues and partially released into the plasma. Using four immunostaining techniques, we have evaluated the question of whether dentin Gla proteins (DGP) are transported to the mineralization front through the odontoblast processes. Undecalcified sections of rat incisors and molar tooth germs were immunostained with affinity-purified antibodies to DGP using the following methods: indirect immunofluorescence; peroxidase-antiperoxidase (PAP); avidin-biotin-peroxidase complex (ABC-peroxidase); and avidin-biotin-gold complex with silver enhancement (ABC-GSS). The results obtained with these four procedures were compared with respect to the developmental appearance of DGP, staining intensity and presence in odontoblastic processes, predentin, dentin, and blood vessels. Qualitatively, similar results were obtained with the four, with respect to the distribution and developmental appearance of DGP, with two exceptions: indirect immunofluorescence never stained DGP within blood vessels, whereas the other methods occasionally did; and because of its sensitivity, only the ABC-GSS method revealed immunostaining for DGP in odontoblastic processes. All methods revealed weak immunostaining in predentin which was considerably enhanced with hyaluronidase treatment; however, hyaluronidase only moderately increased predentin immunostaining with ABC-GSS. Of these four procedures, ABC-GSS is the most sensitive; however, ABC-GSS appears to detect predominantly antigens at the surface of tissue sections. We conclude that DGP is present in odontoblastic processes but in low amounts; the weak staining was due either to rapid transport of DGP through the process or to the fact that this mode of transport is limited.

scholarly journals Detection of Streptococcus Suis by in Situ Hybridization, Indirect Immunofluorescence, and Peroxidase-Antiperoxidase Assays in Formalin-Fixed, Paraffin-Embedded Tissue Sections from Pigs

2000 ◽  
Vol 12 (3) ◽  
pp. 224-232 ◽  
Author(s):  
Mette Boye ◽  
Anne A. Feenstra ◽  
Conny Tegtmeier ◽  
Lars Ole Andresen ◽  
Søren R. Rasmussen ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Indirect Immunofluorescence ◽  
Polyclonal Antibodies ◽  
Single Cells ◽  
Streptococcus Suis ◽  
Tissue Sections ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Streptococcus suis is an important pathogen in pigs and is considered a zoonotic agent. To aid diagnosis of infection caused by S. suis, a species-specific probe targeting 16S ribosomal RNA was designed and used for fluorescent in situ hybridization. Two additional immunohistochemical detection methods, an indirect immunofluorescence assay and a peroxidase-antiperoxidase method, using polyclonal antibodies also were developed. The specificity of the oligonucleotide probe was examined by whole-cell and dot-blot hybridization against reference strains of the 35 serotypes of S. suis and other closely related streptococci and other bacteria commonly isolated from pigs. The probe was specific for S. suis serotypes 1–31. The specificity of the polyclonal antibodies, which has previously been evaluated for use in diagnostic bacteriology for typing of serotype 2, was further evaluated in experimentally infected murine tissue with pure culture of different serotypes of S. suis, related streptococci, and other bacteria commonly found in pigs. The polyclonal antibodies against S. suis serotype 2 cross-reacted with serotypes 1 and 1/2 in these assays. The in situ hybridization and the immunohistochemical methods were used for detection of S. suis in formalin-fixed, paraffin-embedded tissue sections of brain, endocardium, and lung from pigs infected with S. suis. The methods developed were able to detect single cells of S. suis in situ in the respective samples, whereas no signal was observed from control tissue sections that contained organisms other than S. suis. These techniques are suitable for determining the in vivo localization of S. suis for research and diagnostic purposes.

scholarly journals FRET–CLSM and double-labeling indirect immunofluorescence to detect close association of proteins in tissue sections

2006 ◽  
Vol 86 (8) ◽  
pp. 853-864 ◽  
Author(s):  
Peter König ◽  
Gabriela Krasteva ◽  
Claudia Tag ◽  
Inke R König ◽  
Christoph Arens ◽  
...  
Keyword(s):  
Indirect Immunofluorescence ◽  
Close Association ◽  
Double Labeling ◽  
Tissue Sections

scholarly journals Multiple Iterative Labeling by Antibody Neodeposition (MILAN)

2019 ◽  
Author(s):  
Giorgio Cattoretti ◽  
Francesca Maria Bosisio ◽  
Lukas Marcelis ◽  
Maddalena Maria Bolognesi
Keyword(s):  
Image Analysis ◽  
Tissue Section ◽  
Indirect Immunofluorescence ◽  
Analysis Software ◽  
Tissue Sections ◽  
Image Analysis Software ◽  
Single Section ◽  
Fluorescent Image ◽  
Secondary Antibodies

Abstract Multiplexing, labeling for multiple immunostains the very same cell or tissue section in situ, is of considerable interest. The major obstacles to the diffusion of this technique are high costs in custom antibodies and instruments, low throughput, scarcity of specialized skills or facilities. We have validated and detail here a method based on common primary and secondary antibodies, diffusely available fluorescent image scanners and routinely processed tissue sections \(FFPE). It entails rounds of four-color indirect immunofluorescence, image acquisition and removal \(stripping) of the antibodies, before another stain is applied. The images are digitally registered and the autofluorescence is subtracted. Removal of antibodies is accomplished by disulphide cleavage. In excess of 50 different antibody stains can be applied to one single section from routinely fixed and embedded tissue. This method requires a modest investment in hardware and materials and uses freeware image analysis software.

scholarly journals Multiple Iterative Labeling by Antibody Neodeposition (MILAN)

2019 ◽  
Author(s):  
Giorgio Cattoretti ◽  
Francesca Maria Bosisio ◽  
Lukas Marcelis ◽  
Maddalena Maria Bolognesi
Keyword(s):  
Image Analysis ◽  
Tissue Section ◽  
Indirect Immunofluorescence ◽  
Analysis Software ◽  
Tissue Sections ◽  
Image Analysis Software ◽  
Single Section ◽  
Fluorescent Image ◽  
Secondary Antibodies

Abstract (What’s new in protocol Version 5: an expanded troubleshooting section, more validated antibodies)Multiplexing, labeling for multiple immunostains the very same cell or tissue section in situ, is of considerable interest. The major obstacles to the diffusion of this technique are high costs in custom antibodies and instruments, low throughput, scarcity of specialized skills or facilities. We have validated and detail here a method based on common primary and secondary antibodies, diffusely available fluorescent image scanners and routinely processed tissue sections \(FFPE). It entails rounds of four-color indirect immunofluorescence, image acquisition and removal \(stripping) of the antibodies, before another stain is applied. The images are digitally registered and the autofluorescence is subtracted. Removal of antibodies is accomplished by disulphide cleavage. In excess of 50 different antibody stains can be applied to one single section from routinely fixed and embedded tissue. This method requires a modest investment in hardware and materials and uses freeware image analysis software.

scholarly journals Diagnosis of Statin-Induced Necrotizing Myopathy: Contribution of Anti-HMGCR Antibodies

2021 ◽  
Vol 34 (13) ◽  
Author(s):  
Cláudia Fidalgo ◽  
Alice Mendes ◽  
Rosário Cunha ◽  
Fernando Rodrigues
Keyword(s):  
Muscle Biopsy ◽  
Indirect Immunofluorescence ◽  
Gold Standard ◽  
Case Reports ◽  
Antibody Detection ◽  
Fluorescence Pattern ◽  
Tissue Sections ◽  
Necrotizing Myopathy ◽  
Immune Mediated ◽  
Essential Contribution

Over the last few years, several cases of statin-induced necrotizing myopathy have been described. This myopathy is characterized by the necrosis of muscle fibers and the presence of anti-3-hydroxy-3-methylglutaryl-coenzyme A reductase (anti-HMGCR) antibodies. Although the diagnosis of myopathies relies on muscle biopsy, which is considered the gold-standard, the search for autoantibodies has proved to be an essential contribution to the diagnosis of immune-mediated myopathies. The detection of anti-HMGCR antibodies in the patient’s serum can be performed by enzyme immunoassays, and more recently, by imunofluorescence. As for the latter, the detection of anti-HMGCR antibodies is performed on tissue sections by indirect immunofluorescence and is characterized by a typical fluorescence pattern called “HMGCR Associated Liver IFL Pattern”. The authors present two case reports that show the importance of diagnosing statin-induced necrotizing myopathy as quickly as possible and the contribution of anti-HMGCR antibody detection for the diagnosis.

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