Junqueira Ch1
Junqueira Ch1
Junqueira Ch1
Junqueira Ch1
Junqueira Ch1
Junqueira Ch1
Junqueira Ch1
Junqueira Ch1
Junqueira Ch1
Junqueira Ch1
Junqueira Ch1
Junqueira Ch1
Junqueira Ch1
Junqueira Ch1
Junqueira Ch1
Junqueira Ch1
Junqueira Ch1
Junqueira Ch1
Junqueira Ch1
Junqueira Ch1
The paraffin H&E section is the standard preparation behind more than nine out of every ten histology slides made worldwide, and it is the first “workflow” question students are expected to answer cold. The reason this question is so common is that every later technique — immunohistochemistry, special stains, even some EM protocols — sits on top of this same sequence. Learning the order is not just memorisation; each step exists to undo a problem created by the previous step, and that logic is what locks the order in.
1. Fixation
The tissue is immersed in 10% neutral-buffered formalin as soon as possible after removal. Formaldehyde cross-links protein amino groups, killing every cell instantly, halting autolytic enzymes and bacterial putrefaction, and hardening the tissue enough to handle. The neutral buffer prevents acidic formalin-pigment deposits. Tissue blocks should be no more than ~5 mm thick so that the fixative penetrates fully within hours.
2. Dehydration
Paraffin is hydrophobic, so the water that fills living tissue must be removed before wax can infiltrate it. The block is passed through a series of graded ethanols (typically 50% → 70% → 95% → 100%), each for a defined time. A sudden jump straight into absolute alcohol would shrink and distort the specimen; the gradient is gentle.
3. Clearing
Ethanol and molten paraffin are not miscible either, so a third reagent is needed to bridge them. Xylene (or, in some labs, toluene) is miscible with both ethanol and wax. It replaces the alcohol in the tissue and also renders the specimen translucent — hence the name “clearing.”
4. Embedding
The cleared tissue is placed in molten paraffin wax (~58°C). The wax infiltrates the spaces formerly held by water and is then cooled in a mould to form a hard, supportive block from which thin sections can be cut without crushing.
5. Sectioning
The block is trimmed and mounted on a microtome, which advances the block by a precise micrometre amount between each pass of a steel knife. Routine sections are 5–7 µm thick — a ribbon of paraffin-embedded tissue that is floated out on a warm water bath to flatten and then picked up onto a glass slide.
6. Staining and mounting
The slide is reverse-engineered for staining: xylene removes the wax (de-waxing), graded alcohols then water bring it back into aqueous solution (rehydration), the H&E stains are applied in turn, the slide is dehydrated and cleared again, and finally a coverslip is sealed on with mountant to preserve the section indefinitely.
Clinical anchor
This whole sequence takes most of a day, which is why frozen sections exist as a faster alternative for intra-operative diagnosis. But for the millions of biopsies reported every year — from skin lesions to tumour resections to renal biopsies — this paraffin pipeline is what produces the slide the pathologist actually looks at.
The light microscope and the transmission electron microscope are not rival instruments — they are complementary, asking different questions of the same tissue. The LM tells you which organ, which tissue, which cell type. The TEM tells you which organelles a cell contains and how its membranes are arranged. A complete histological understanding always comes from putting the two together. This question is testing whether you can lay them side by side along the same five or six axes.
Illumination and lenses
The LM passes a beam of visible light through the specimen and focuses it with glass lenses. The TEM substitutes a focused beam of electrons for light, and electromagnetic coils for the lenses. Because the column must be a vacuum (electrons would scatter off air molecules), the whole instrument is much larger, more expensive and slower to use.
Resolution
The decisive difference. Resolution is limited by wavelength: visible light bottoms out around 0.2 µm (200 nm) — just good enough to see cells and large organelles like nuclei. Electrons have a wavelength roughly a thousand times shorter, so the TEM resolves to about 0.1–0.2 nm. This is what made organelles visible for the first time in the 1950s — RER, Golgi cisternae, ribosomes, junctional complexes.
Fixation and processing
LM tissue is fixed in 10% neutral-buffered formalin, then dehydrated, cleared and embedded in paraffin wax. TEM tissue is fixed in glutaraldehyde (a much stronger protein cross-linker) and post-fixed in osmium tetroxide, which also fixes lipids and gives membrane contrast; it is then embedded in epoxy resin, which is hard enough for ultrathin sections.
Sectioning
The LM section is cut on a microtome with a steel knife at 5–10 µm. The TEM section is cut on an ultramicrotome with a diamond (or freshly broken glass) knife at 50–100 nm — some forty to a hundred times thinner — thin enough for an electron beam to pass through.
Contrast and image
LM contrast comes from chemical dyes (H&E, PAS, silver, trichrome) that produce real colour. TEM contrast comes from heavy metals (osmium on lipids, uranyl acetate on nucleic acids, lead citrate on membranes), and the resulting image is greyscale only: areas that scatter many electrons appear electron-dense (dark), areas that scatter few appear electron-lucent (light). There is no colour in EM.
Clinical anchor
Diagnostic surgical pathology is overwhelmingly an LM speciality — H&E plus a panel of special stains and immunohistochemistry. TEM still has dedicated diagnostic uses where ultrastructure is the answer: renal biopsy (podocyte foot processes, electron-dense immune deposits and basement-membrane thickness in glomerular disease), ciliary disorders (dynein-arm defects in primary ciliary dyskinesia), and the identification of viral particles or amyloid fibrils when other methods fall short.
Of all the questions in this unit, this is the one that matters most for the rest of the year. Every later organ slide you describe will use the two words basophilic and acidophilic, and every later cell you identify will be classified by the colours its parts take up. Once you can explain the chemistry behind these two terms — and resist the common backwards-naming trap — the rest of histology unlocks.
The H&E stain
H&E is shorthand for haematoxylin and eosin, two dyes of opposite charge applied in sequence to almost every routine slide. Haematoxylin (technically applied as a metal–dye complex, the haemalum) acts as a basic, positively-charged dye. Eosin is an acidic, negatively-charged dye. The two together generate the universal blue-and-pink slide that defines histology in textbooks, lectures and exam halls.
The chemistry of binding
Dyes bind tissue components by ionic attraction. The basic dye haematoxylin (positive) sticks to the negatively-charged groups in tissue — chiefly the phosphate backbone of nucleic acids and the sulphate/carboxyl groups of acidic glycosaminoglycans. The acidic dye eosin (negative) sticks to positively-charged amino groups on cytoplasmic and matrix proteins. The colour pattern of any cell is therefore a chemical map of where the charge lives.
Basophilia — loving the base
A structure is called basophilic when it has high affinity for the basic dye and therefore stains blue-purple. The naming convention is counter-intuitive: a basophilic structure is one that binds the basic dye, which means the structure itself is acidic. Classic basophilic structures include the nucleus (DNA phosphate), the nucleolus (rRNA), rough endoplasmic reticulum and free ribosomes (rRNA), cartilage matrix (chondroitin sulphate), and the granules of basophils and mast cells. When you see a cell whose cytoplasm is strongly basophilic — a plasma cell, a pancreatic acinar cell, the Nissl bodies of a neuron — the blue colour is telling you the cytoplasm is packed with ribosomes, and that cell is busy making protein for export.
Acidophilia (eosinophilia) — loving the acid
A structure is acidophilic (synonym eosinophilic) when it binds the acidic dye eosin and stains pink-red. These structures are themselves basic — rich in cationic amino-acid side chains. The list is long and important: most cytoplasm, collagen fibres, mitochondria, red blood cells, keratin, and the secretory zymogen granules of many exocrine cells. The granules of the eosinophil leukocyte are themselves intensely eosinophilic — which is how the cell got its name.
Clinical anchor
The pathologist grading a cancer looks at the same blue and pink. Tumour nuclei are typically large, irregular and hyperchromatic — intensely basophilic because of dense, disordered chromatin — with a high nucleus-to-cytoplasm ratio. In chronic inflammation, the strongly basophilic cytoplasm of plasma cells (full of antibody-making RER) is the give-away of an ongoing humoral response. Two dyes, the entire diagnostic vocabulary of microscopy.
H&E is the workhorse, but it is essentially blind to several structures that matter enormously for diagnosis: reticular fibres melt into the background, basement membranes are barely visible, glycogen is washed out by processing, elastic fibres look like nothing, and amyloid is undetectable. For each of these gaps the histologist reaches for a special stain — a chemistry tuned to one target. Master the small standard set and the same handful of stains will reappear in every organ unit for the rest of the year.
PAS (Periodic acid–Schiff)
Periodic acid oxidises the 1,2-glycol groups on tissue carbohydrates to aldehydes, and Schiff reagent then binds the aldehydes to produce a magenta colour. PAS lights up glycogen (hepatocytes, striated muscle), basement membranes (kidney glomeruli, epithelial linings), mucin in goblet cells, the brush border (glycocalyx) of the intestine, and fungal cell walls. It is the standard stain for showing “sugar.”
Silver impregnation
Silver salts bind to certain tissue components and are reduced to elemental silver, depositing as fine black threads against a pale background. The principal targets are reticular fibres (type III collagen) — the supporting meshwork of liver, lymph node, spleen and bone marrow — together with basement membranes, nerve fibres and the neurofibrils inside neurons. Structures that take silver this way are called argyrophilic.
Masson trichrome
The “three-colour” stain separates collagen (blue or green, depending on the variant) from muscle and other cytoplasm (red) and nuclei (dark). It is the standard tool for assessing fibrosis — how much collagen scar tissue has replaced functional parenchyma. Cirrhotic liver, post-infarct heart and fibrotic lung are all trichrome territory.
Elastic stains (orcein, resorcin-fuchsin, Verhoeff)
Elastic fibres are essentially invisible on H&E. Orcein, resorcin-fuchsin and Verhoeff's haematoxylin all selectively stain elastic fibres and laminae brown, purple or black. Use them in the elastic arteries (aorta and its branches), in elastic cartilage of the ear, and in lung tissue.
Toluidine blue and metachromasia
Toluidine blue is a blue dye that turns purple-red when it binds densely sulphated GAGs — the property called metachromasia. It is the standard way to highlight mast-cell granules (heparin) and cartilage matrix (chondroitin sulphate).
Stains for fat (frozen sections)
Because alcohol and xylene dissolve fat out of paraffin sections, lipids must be studied on frozen sections. Sudan dyes (Sudan III, IV, Sudan Black) stain neutral fat orange-red or black. Osmium tetroxide blackens fat by binding to membrane lipids.
Clinical anchor
In a renal biopsy, four stains are usually run side by side: H&E for general architecture, PAS to show basement-membrane thickness, silver (Jones methenamine) for reticular meshwork and BM spikes, and trichrome to assess interstitial fibrosis. The combination tells the pathologist whether a glomerular disease is membranous, mesangial, sclerosing or interstitial — and therefore how it should be treated.
Immunohistochemistry (IHC) is the technique that changed twentieth-century pathology more than any other. Where routine stains show only structure or broad chemistry classes, IHC localises one specific molecule — one protein, one antigen — exactly where it sits in the tissue. It is the basis of modern tumour classification and increasingly of treatment selection, and every medical graduate is expected to know its principle, its workflow and its clinical reach.
Principle — molecular recognition made visible
The principle is the antigen–antibody reaction borrowed straight from immunology. An antibody raised against a defined antigen will bind that antigen with high specificity. If you attach a visible label to the antibody — an enzyme that develops a coloured product, or a fluorochrome that glows under UV light — the location of the antibody, and therefore of its antigen, is marked exactly on the slide. The technique converts a question of chemistry (is this protein here?) into a visual answer (look, brown signal in these cells).
Method — the indirect protocol
The standard workflow is the indirect two-step method. A section is dewaxed and rehydrated as usual, and pretreatment steps (often heat-induced antigen retrieval) expose the epitope that fixation has masked. An unlabelled primary antibody specific for the target antigen is applied and allowed to bind, then washed off. A labelled secondary antibody directed against the species of the primary is applied; several secondary antibodies bind each primary, which amplifies the signal. The label is then developed: a peroxidase enzyme converts DAB chromogen to an insoluble brown product visible under the light microscope, or a fluorochrome (FITC green, rhodamine red) is viewed under a fluorescence microscope. The slide is finally counter-stained (usually with haematoxylin) so that the structural context is visible alongside the antigen signal.
Uses — diagnostic pathology and beyond
The clinical impact is enormous. In an undifferentiated tumour, IHC tells the pathologist what kind of cell the cancer arose from: cytokeratin for epithelial origin (carcinoma), vimentin for mesenchymal origin (sarcoma), CD45 (LCA) for lymphoid origin (lymphoma), and S-100/HMB-45 for melanocytic origin (melanoma). In lymphoma it sub-types further: CD20 for B-cells, CD3 for T-cells. In breast cancer the IHC panel of oestrogen receptor, progesterone receptor and HER2 directly decides whether the patient receives tamoxifen, an aromatase inhibitor or trastuzumab. Beyond cancer, IHC localises infectious organisms (CMV, herpes), defines basement-membrane components in renal disease, and underlies vast amounts of research in cell biology and neuroscience.
Clinical anchor
A woman presents with a breast lump. The biopsy is reported as invasive ductal carcinoma on H&E. The same block is then stained by IHC for ER, PR and HER2. Strong nuclear ER and PR positivity tells the oncologist that hormonal therapy will work; HER2 over-expression (with confirmatory FISH if equivocal) tells them that trastuzumab will too. The treatment plan is built directly on the IHC slide — structure plus specific molecule, the technique that ties microscope to molecule.