practical 1- H&E staining Flashcards
what governs the overall staining pattern
rate of which a dye is taken up by a tissue component or the rate of loss of due from a tissue component
what are the 3 layers composing the skin
epidermis dermis subcutis (hypodermis)
In H&E staining, how do we differentiate a section after staining with harris’s haematoxylin and why do we do this
differentiate in 1% acid alcohol for 2-5 seconds use of acidic solutions to remove excess background staining
In H&E staining, what step is taken after dewaxing, hydrating, haematoxylin staining, differentiating and blueing
immerse in eosin for 2-4 minutes
in a rate depending staining process, what is a progressive dye
dyes staining more intensely the longer the tissue remains in the dye bath
what colour stain does eosin produce
red cytoplasmic stain
where might you see cells in the process of dividing (mitosis)
mucosa- base of intestinal crypts, this is to replenish cells at the epithelium
In H&E staining, what blueing reagent is used and why do we use it?
Scotts solution necessary to convert nuclear colouration from reddish purple to crisp blue/purple enhances the contrast of the H&E stain by increasing the crispness of the haematoxylin
what is meant by basophilia
tissues with affinity for haematoxylin (basic dye) is base loving
what is the function of the muscularis externa
undergoes segmentation contractions (which mixes chyme in the small intestine) and weak peristaltic contraction (moves chyme along in the small intestine)
what does harris’s haematoxylin use to alter the specificity of its stain explain the procedure
uses aluminium salts (haemalum) as a mordant rinse slide in tap water to blue the section haematoxylin used regressively with controlled differentiation in acid alcohol reblueing in tap water (must be alkaline)
how many layers of cells are there in the absorptive epithelium proper
mucosa- one layer
what is the function of the submucosa
contains brunners galnds, they secrete alkaline mucus which neutralises gastric acid in chyme
in a rate depending staining process, what is a regressive dye
a dye may be selectively lost/removed fro a tissue to distiniguish/differentiate between components with stronger or weaker affinities for the dye
how is haematoxylin produced
extracted in water and precipitated with urea
what type of dye are the following Ehrilchs, Mayers, Harris, Gills, Coles, Delafield, Carazzis
haematoxylin dyes
what type of a staining procedure is H&E staining
regressive
what is the function of enteroendocrine cells and paneth cells
enteroendocrine cells secrete hormones (somatostatin) paneth cells have an immune cell function
what is eosin and how does it work
acidic dye, but binds to basic (eosinophillic) compounds/structures
see the skin diagram in the test booklet, label A-L
A- stratium corneum (cornified/horny layer) B- pickle cell layer C- dermis D- fat E- sweat gland F- hair follicle G- blood vessels H- sebaceous (oil) gland I- lymph vessel J- sensory nerve receptor K- hair L- pores
In H&E staining, how do we dewax a section
place in histoclear for 5 minutes
what is meant by acidophilia
tissues with affinity for eosin (acidic dye) is acid loving
look at tissue A in the test booklet, label A, B and C how are the columns of hepatocytes that radiate from the central vein seperated
A- portal triad (proper hepatic artery, hepatic portal vein and bile duct) B- hepatic vein C- hexagonal hepatic lobule (bile canaliculi, sinusoidal capillaries, hepatocytes) seperated by canniculae of the bile ducts
In H&E staining, what do we do after dewaxing and hydrating a sample
immerse in Harris’s haematoxylin for 5 minutes
what alters specificity of a haematoxylin stain
use of a mordant (inorganic stain-fixing solution)
what is the most common method used to enhance contrast between cell structures and tissues
dyes e.g. Haematoxylin and Eosin staining
where does haematoxylin come from
natural product made from haematoxylum campechianum (logwood)
what are the 3 zones in the duodenum state what each zone consists of
mucosa- inner absorptive epithelium with associated connective tissue (such as blood vessels) consists of lamina propria and muscularis mucosa submucosa- underlying zone of loos CT muscularis externa- outer muscle coat, consists of circular and longitudinal layers
what is meant by eosinophilia
eosin loving, dye term used to describe tissue affinity
what colour staining does H&E produce
red/blue
what procedure does H&E staining rely on
acid-base dye interactions
what is the function and structure of the subcutis (hypodermis)
layer containing main vascular supply contains adipose tissue and fibrous tissue
what is the function and structure of the epidermis name the layers, inner to outer
upper, protective zone comprising several sublayers of stratified squamous epithelium boys smell girls like chocolate basale, spinosum, granulosum, corneum
what are the different types of eosin stains
eosin Y and eosin B
In H&E staining, how do we dehydrate a sample
by passing through alcohols of increasing concentration 70% alcohol for 1 minute 90% alcohol for 1 minute absolution (100%) alcohol for 1 minute
what is the function and structure of the dermis
underlying zone containing several glands and structures which include: hair follicles, sebaceous glands, eccrine sweat glands and apocrine glands
look at the diagram of tissue C- duodenum on the test booklet and label A-G
A- epithelial layer of cells B- villus C- lamina propria D- muscularis mucosae (mucosa) E- intestinal crypts (crypt of lieberkuhn) F- brunners gland G- serosa (outer layer)
how do goblet cells and enterocytes differ
goblet cells are cup shaped (involution doesnt stain)- secrete mucus enterocytes are rectangular and have an elongated nuclei
what is meant by dye-tissue affinity in H&E staining
measure of a dye to transfer from a dye bath onto a tissue section
how is the muscularis externa arranged
outer thinner layer has longitudinal arrangement inner thicker layer has circular arrangement
put the following duodenum layers in order (inner to outer) lamina propria sub mucosa adventitia epithelium muscularis propria muscularis mucosa
epithelium lamina propria muscularis mucosa sub mucosa muscularis propria adventitia
name 3 other methods used to enhance contrast between cell structures and tissues
polarised light, dark field, phase contrast
how thin must tissue sections be when viewing through a light microscope
<15µm
what process must a haematoxylin dye undergo before use as a stain
requires oxidation to haematein
In H&E staining, how is the sample cleared at the end
immersed in histoclear for 5 minutes and mounted in DePeX
what does a haematoxylin dye stain, give examples
basophillic structures such as nucleic acids, ribosomes
In H&E staining, how do we hydrate a sample
by passing through alcohols of decreasing concentration 100% (absolute) alcohol for 1 minute 90% alcohol for 1 minute 70% alcohol for 1 minute
what is eosin derived from
fluorescein (fluoresces)
