What is paraffin immunohistochemistry?
Immunohistochemistry (or IHC) is a method for demonstrating the presence and location of proteins in tissue sections. Though less sensitive quantitatively than immunoassays such as Western blotting or ELISA, it enables the observation of processes in the context of intact tissue.
How do you stain paraffin-embedded tissue?
Immerse the tissue in 70% ethanol three times for 30 minutes each at room temperature. Immerse the tissue in 90% ethanol two times for 30 minutes each at room temperature. Immerse the tissue in 100% ethanol three times for 30 minutes each at room temperature.
Can you do immunofluorescence on paraffin-embedded sections?
IHC can be applied on tissues prepared either through paraffin embedding or through cryopreservation. Despite its wide application, inexperienced researchers often face trouble in successfully detecting and analyzing a clear fluorescent signal of their protein of interest.
How do you write an IHC protocol?
Immunohistochemistry Protocol
- Immerse the slides in xylene (mixed isomers) 2 times for 10 minutes each.
- Immerse the slides in 100% alcohol 2 times for 10 minutes each.
- Immerse the slides in 95% alcohol for 5 minutes.
- Immerse the slides in 70% alcohol for 5 minutes.
- Immerse the slides in 50% alcohol for 5 minutes.
What is paraffin section?
Paraffin sectioning is the procedure of cutting thin slices of tissue that has been dehydrated and infiltrated with wax using specialized equipment. This tissue is then embedded in wax before being cut on a microtome.
What are paraffin blocks?
Paraffin-embedded tissue blocks usually refer to formalin fixed paraffin embedded (FFPE) tissue specimens that have been used in various therapeutic applications and research for many decades.
How are paraffin sections prepared in the lab?
Sectioning Protocol: Section paraffin blocks at the desired thickness (usually 4-5 µm) on a microtome and float on a 40°C water bath containing distilled water. Transfer the sections onto a Superfrost Plus slide. Allow the slides to dry overnight and store slides at room temperature until ready for use.
What is the thickness of paraffin embedded tissue sections?
Section thickness Paraffin sections for light microscopy are typically 5 μm thick. Paraffin wax does not provide a sufficiently hard matrix for cutting thinner slices needed for electron microscopy, typically 80-100 nm thick.
What are main steps in immunohistochemistry procedure?
A general immunohistochemistry protocol consists of four main steps:
- Fixation—to keep everything in its place.
- Antigen retrieval—to increase the availability of proteins for detection.
- Blocking—to minimize pesky background signals.
- Antibody labeling and visualization—to get the pretty pictures.
Why is paraffin used in histology?
What is the paraffin technique? In this technique, tissues are fixed, and embedded in wax. This makes the tissue hard, and much easier to cut sections from.
How do you do paraffin sections?
- Chill paraffin-embedded tissue blocks on ice before sectioning.
- Fill a waterbath with ultrapure water and heat to 40-45oC.
- Place the blade in the holder, ensure it is secure and set the clearance angle.
- Insert the paraffin block and orientate so the blade will cut straight across the block.