Developmental Lab Quiz

Immunohistochemistry purpose

A method to detect specific proteins in tissues using antibodies and visualize where those proteins are located.

What phospho-histone H3 marks

Cells actively undergoing mitosis, specifically during the G2/M transition.

Why we use planaria for this stain

Only neoblasts divide in planaria, so phospho-H3 stain highlights stem cells involved in regeneration.

Why remove mucus layer

Planaria produce a mucus coat that blocks antibodies; the HCl wash removes it so staining works.

Day 1 HCl wash

Fragments are placed in 2% HCl on ice and shaken to kill and de-mucus the tissue quickly.

Purpose of fixation

Fixation preserves the tissue in its current state so protein localization remains accurate.

Carnoy’s fixative step

After HCl removal, tissue is placed in Carnoy’s (ethanol-based fixative) to “freeze” cellular structure.

Methanol rinse

Rinses off fixative and prepares tissue for bleaching and later staining steps.

Purpose of bleaching

Removes the planarian’s natural brown pigment, allowing fluorescent signal to be visible.

Bleaching solution contents

Hydrogen peroxide + formamide + PBSTx under UV light.

What PBSTx does

A detergent solution that permeabilizes cells and washes away unbound antibodies.

Purpose of blocking step

A BSA protein-rich solution prevents antibodies from sticking nonspecifically and causing background noise.

Day 2 blocking procedure

Tissue is incubated in 1% BSA for 1 hour at room temperature.

Primary antibody used

Rabbit anti-phospho-H3 at 1:500, labeling mitotic cells.

Why primary antibody incubates overnight

Ensures full penetration of antibody through thick planarian tissues.

Day 3 rinse of primary antibody

Quick PBSTx rinses followed by a wash to remove excess unbound primary.

Secondary antibody purpose

Binds to the primary antibody and carries the enzyme that allows fluorescence to develop.

Secondary antibody used

Goat anti-rabbit HRP at 1:1000, incubated overnight at 4°C.

What HRP does

HRP enzyme activates the fluorescent tyramide substrate, creating a bright signal where antibody is bound.

Purpose of tyramide

Amplifies signal by depositing fluorescent molecules at antibody binding sites.

Day 4 substrate addition

PBSTx is replaced with 488-tyramide to prepare for HRP activation.

Why add hydrogen peroxide

Hydrogen peroxide activates HRP so it begins the tyramide reaction.

Day 4 reaction time

10 minutes pre-incubation with substrate, then 30 minutes with hydrogen peroxide.

Final rinses

Two quick PBSTx rinses, then two 15-minute washes to stop the reaction and remove leftover reagents.

What green fluorescence indicates

Cells currently in mitosis — the neoblasts actively dividing.

Why use secondary instead of direct fluorescent primary

Secondary antibodies amplify signal and provide stronger, clearer staining.

Reason for so many washes

To remove unbound antibodies, reduce background, and ensure only true signal remains.

Overall workflow summary

Remove mucus -> fix tissue -> bleach pigment -> permeabilize -> block -> primary antibody -> wash -> secondary antibody -> develop stain -> wash -> image.

What this lab teaches

How to visualize dividing cells, handle antibody staining, and understand tissue processing in histology.