Jul 22, 2025
Cytochemical staining of endocytosed HRP, flat embedding
- Merja Joensuu1
- 1The University of Queensland
- Neuronal b-oxidation
Protocol Citation: Merja Joensuu 2025. Cytochemical staining of endocytosed HRP, flat embedding. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw4599lmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 22, 2025
Last Modified: July 22, 2025
Protocol Integer ID: 222962
Keywords: HRP, Horse radish peroxidase, Cytochemical staining, Synaptic vesicles, Endosomes, Neurons, cytochemical staining of endocytosed hrp, horseradish peroxidase, endocytic compartments such as synaptic vesicle, endocytosed hrp, synaptic vesicle, cytochemical staining, endocytic compartment, diaminobenzidine cytochemistry, endosome, neuron
Abstract
In this protocol, neurons are pulse-chased with horseradish peroxidase (HRP) to induce activity-dependent uptake of HRP into endocytic compartments such as synaptic vesicles and endosomes. HRP is visualised via diaminobenzidine cytochemistry followed by osmium tetroxide, producing a dark brown, electron-dense precipitate within labeled structures. After staining, neurons are flat-embedded in LX112 directly on the plastic culture dish.
Materials
• Horse radish peroxidase (HRP)
• 2% Paraformaldehyde
• 1.5% Glutaraldehyde
• 0.1 M Sodium cacodylate (NaCac) buffer, pH 7.4
• 50 mM Tris buffer, pH 7.6
• 10 mg DAB tablet
• 30% H₂O₂
• 1% OsO₄
• 6% potassium ferrocyanide
• 2 mL Mq (Milli-Q water)
• 4% OsO₄
• 2% UA (uranyl acetate, aq)
• Ethanol (EtOH) series: 50%, 60%, 70%, 90%, 100%
• LX112 resin
• Beam capsules
• Equipment: microwave (MW), vacuum (VAC), ultramicrotome (e.g., Leica Biosystems, UC6FCS)
Troubleshooting
HRP-labelling of endocytic compartments
35m
For high potassium neuronal depolarisation, stimulate neurons in prewarmed (37°C) high K⁺ buffer supplemented with 10 mg/ ml horse radish peroxidase (HRP) and incubate cell incubator at for 5 minutes at 37°C with 5% CO₂. For electric field stimulation assays, add 1 mg/ml HRP diluted in prewarmed (37°C) low K+ buffer for the duration of high frequency stimulation (300 APs at 50 Hz for 6 s).
5m
High K+ buffer consists of 0.5 mM MgCl₂ (Chem-Supply, MA029), 2.2 mM CaCl₂ (Sigma-Aldrich, C5080), 56 mM KCl (Ajax Finechem, 1206119), 95 mM NaCl(Amresco, X190), 5.6 mM D-glucose (Amresco, 0188), 0.5 mM ascorbic acid (Sigma-Aldrich, A5960), 0.1% BSA (Sigma-Aldrich,
A7638), 15 mM HEPES (Gibco, 15630-080), pH 7.4, 290–310 mOsm.
Low K⁺ buffer consists of 0.5 mM MgCl₂ (Chem-Supply, MA029), 2.2 mM CaCl₂ (Sigma-Aldrich,
C5080), 5.6 mM KCl (Ajax Finechem, 1206119), 145 mM NaCl (Amresco, X190),
5.6 mM D-glucose (Amresco, 0188), 0.5 mM ascorbic acid (Sigma-Aldrich, A5960),
0.1% BSA (Sigma-Aldrich, A7638), 15 mM HEPES (Gibco, 15630-080), pH 7.4
290–310 mOsm
Wash away all free HRP from the plates using prewarmed (37°C) low K⁺ buffer.
Chase the HRP uptake in neurons for 10 or 30 minutes in low K⁺ buffer in a cell incubator (37°C with 5% CO₂).
30m
Cytochemical staining of HRP
50m
Fix cells with 2% Paraformaldehyde (Electron Microscopy Sciences, 15710), 1.5% Glutaraldehyde (Electron Microscopy Sciences, 16210) in 0.1 M Sodium cacodylate (NaCac, Sigma-Aldrich, C0250) buffer pH 7.4 for 20 minutes at room temperature. NOTE! Use EM-grade, fresh, fixatives only and perform the liquid exchange in a fume hood.
20m
Wash 2 times with 0.1 M NaCac buffer, á 3 min. Perform the liquid exchange in a fume hood.
Wash 3 times with 50 mM Tris buffer, pH 7.6, á 5 min. Perform the liquid exchange in a fume hood.
Incubate in DAB solution (with peroxide) for 30 minutes, room temperature, covered from light
- Dilute 10 mg DAB tablet (Sigma-Aldrich, D4418) into 10 ml of 50 mM Tris-buffer, pH 7.6
- Just before use add 7 µl of 30% H₂O₂ to 10 ml of diluted DAB-solution to final concentration of 0.021%.
NOTE: keep DAB solutions covered from light all the time!
30m
Wash 3 times with Tris buffer, á 5 min.
Wash 2 times with 0.1 M NaCac buffer. Perform the liquid exchange in a fume hood.
Embedding monolayers in resin using Biowave (Pelco)
50m
Osmicate using Reduced 1% osmium Tetroxide (OsO₄), 1.5% potassium ferrocyanide (K4[Fe(CN)6]) diluted in Millli-q water. Perform the liquid exchange in a fume hood.
Biowave settings: MW 80 W, 2 min ON – 2 min OFF – 2 min ON, VAC on
Repeat step 10
Water wash:
Biowave settings: MW 250 W, 40 s No VAC. Perform the liquid exchange in a fume hood.
Contrast with 2% uranyl acetate (UA) (aq):
Biowave settings: MW 150 W, 1 min ON – 1 min OFF – 1 min ON, VAC on (optional). Perform the liquid exchange in a fume hood.
Dehydration in a fume hood (no Biowave usage):
- 50% EtOH 1min
- 60% EtOH 1min
- 70% EtOH 1min
- 90% EtOH 1min
- Twice: 100% EtOH 1min
1st embedding step: 1:1 (v/v) of 100% EtOH: LX112 resin. Perform the liquid exchange in a fume hood.
Biowave settings: MW 250 W, 3 min VAC on
2st embedding step: 100% LX112. Perform the liquid exchange in a fume hood.
Biowave settings: MW 250 W, 3 min VAC on
3rd embedding step: 100% LX112. Perform the liquid exchange in a fume hood.
Biowave settings: MW 250 W, 3 min VAC on
Place beem capsules filled with 100% LX112 upside down on top of the neurons in a fume hood.
Polymerization at 60°C 48 h
- Samples are polymerized immediately, no need to wait for 2 h
Thin sectioning
Trim 1 mm x 1 mm pyramid, and thin section (80-90 nm) samples using an ultramicrotome (such as Leica Biosystems, UC6FCS).
TEM imaging
For HRP-analysis, TEM images are acquired randomly when HRP signal is observed.
Quantification
The quantification of the number of HRP-containing synaptic vesicles and endosomes is quantified with Adobe Photoshop Count Tool, and the size (sectional area, µm²) of the HRP-stained endosomes is quantified manually using ImageJ/Fiji (https://imagej.nih.gov/ij/)