Histotechnique Overview and Purpose
📌 Histotechnique is a technique for preparing histological slides or preparations of animal or human tissue.
🔬 Its uses include studying the morphology of cells or tissues and detecting active components like enzymes, hormones, or carbohydrates within the tissue.
⚙️ The process involves nine key stages: fixation, sampling, fixation stop point, dehydration, clearing, embedding, sectioning, staining, and photomicrography.

Fixation Solutions Preparation
🧪 Two primary fixative solutions discussed are Bouin's solution and 4% Paraformaldehyde.
💧 Bouin's solution is made just before use by mixing saturated picric acid, formalin, and glacial acetic acid in a 15:5:1 ratio.
🌡️ Paraformaldehyde solution is prepared by dissolving 4% paraformaldehyde in physiological NaCl over a hot plate, followed by adding 2-3 NaOH tablets until clear, and is prepared several days in advance.

Sample Collection (Sampling)
🐭 The animal model used for sampling in this demonstration is the rat.
🔪 Direct sampling method involves anesthetizing the rat (with ketamine and xylazine), dissecting the thorax, quickly removing organs, washing them in physiological NaCl, and placing them in fixative.
💧 The perfusion method involves anesthesia, inserting a needle into the left ventricle connected to an NaCl infusion bag, cutting the right atrium to allow fluid outflow, and then replacing the NaCl bag with one containing paraformaldehyde before organ removal.

Tissue Processing Stages
🕰️ Tissues fixed in Bouin's solution require 24 hours of fixation before being transferred to 70% alcohol (the stopping point), while those fixed in paraformaldehyde require about one week before transfer to 70% alcohol.
🚰 Dehydration removes water from the tissue using ascending concentrations of alcohol: 80%, 90%, and 95% ethanol, followed by three stages of absolute alcohol (1 hour each).
💎 Clearing uses xylene to make the tissue transparent, involving placing the tissue cassette from absolute alcohol into xylene at room temperature for 30 minutes, followed by 30 minutes in an oven at $68^\circ\text{C}$.
🧫 Embedding (Paraffin Infiltration) involves immersing the tissue cassette in liquid paraffin three times (30 minutes each) before molding the tissue block for sectioning.

Sectioning and Staining (H&E)
🔪 Tissue blocks are sectioned using a microtome to a thickness of 4 micrometers ($\mu\text{m}$), and the thin sections are floated on cold water before being mounted onto a glass slide.
🧼 Deparaffinization (removing paraffin) is the first step in staining, achieved by dipping the slide three times in xylene baths, followed by rehydration using descending concentrations of alcohol back to distilled water.
🎨 Hematoxylin staining lasts for 2 minutes, followed by washing, and then Eosin staining for 2 minutes, concluding with a final round of dehydration, clearing, and mounting with a coverslip.

Key Points & Insights
➡️ Histotechnique serves dual purposes: studying morphology and detecting active cellular components like enzymes or hormones.
➡️ The Perfusion Method ensures thorough fixation by circulating fixative directly through the circulatory system, replacing blood with the agent.
➡️ The transition point from fixation to further processing (storage stage) is known as the "stopping point," typically when the tissue is placed in 70% alcohol.
➡️ Final tissue sections are cut very thinly, at $4 \mu\text{m}$, which is critical for light microscopy visibility.

📸 Video summarized with SummaryTube.com on Oct 10, 2025, 12:00 UTC