Sep 17, 2025
Agarose–gelatin double embedding prior to paraffin processing
- Hyoyoung Maeng1,
- Hyuk Song1
- 1School of Advanced Biotechnology, Konkuk University, 120, Neungdong-ro, Gwangjin-gu, Seoul, Republic of Korea, 05029
- BioTechniques
Protocol Citation: Hyoyoung Maeng, Hyuk Song 2025. Agarose–gelatin double embedding prior to paraffin processing. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6z7e5gqe/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: September 16, 2025
Last Modified: September 17, 2025
Protocol Integer ID: 227372
Keywords: Agarose, Gelatin, Double embedding, Neutral buffered formalin, Paraffin, Spheroid, small tissue fragment, Histology, spheroids for routine paraffin, gelatin, paraffin processing, routine paraffin, agarose, paraffin processing this protocol detail, small tissue fragment, handling of small specimen, spheroid
Funders Acknowledgements:
National Research Foundation of Korea
Grant ID: NRF-2022R1A2C1004503
Abstract
This protocol details an agarose–gelatin double embedding workflow to immobilize small tissue fragments or spheroids for routine paraffin embedding. Samples are embedded in a 2% agarose–5% gelatin mixture, then fixed in neutral buffered formalin (NBF) and processed by standard FFPE methods. Compared with conventional methods, it facilitates handling of small specimens, preserves orientation, and minimizes sample damage.
Attachments
Guidelines
Troubleshooting
-Ensure that the agarose–gelatin solution is well mixed and not solidified before embedding.
-Prevent the sample from drying out.
-Process samples in small batches to avoid solidification of the agarose–gelatin gel.
-If the agarose–gelatin gel does not fully cover the sample, add additional solution to embed completely.
Materials
Reagents
AgaroseInvitrogenCatalog #75510-019
Gelatin, from porcine skinMerck MilliporeSigma (Sigma-Aldrich)Catalog #G1890
DPBSSolBioCatalog #DPB 001
Formalin solution, neutral buffered, 10%Merck MilliporeSigma (Sigma-Aldrich)Catalog #HT501128
Materials
200 µL pipet tipCorningCatalog #T-200-Y
1000 µL pipet tipThermo Fisher ScientificCatalog #112NXL-Q
50mL Conical TubeSPL life sciencesCatalog #50050
Petri DishSPL life sciencesCatalog #10090
10mL Serological PipetSPL life sciencesCatalog #91010
MicrotubesCorningCatalog #MCT-175-C
Single Edge BladeDorcoCatalog #DN-52
Kimtech Science WipersYuhan-KimberlyCatalog #41112
Equipment
Equipment
AX1502KR
NAME
ADVENTURER®
BRAND
-
SKU
Equipment
MWO-18M1
NAME
SK Magic
BRAND
-
SKU
Equipment
BW-10E
NAME
Jeiotech
BRAND
-
SKU
Troubleshooting
Safety warnings
-Exercise caution to avoid burns when handling heated gels.
-During fixation, use appropriate equipment to prevent exposure to fixatives.
Before start
-Ensure all reagents and materials are ready before you begin.
-Verify that the samples are properly prepared.
-Confirm the water bath and microwave oven are working properly.
Procedure
Set the water bath to 60 °C .
Prepare a 4 Mass / % volume agarose solution(200 µL per sample) : add agarose powder to distilled water (DW) in a 50 mL conical tube, heat in a microwave oven until fully dissolved.
Keep the 4 Mass / % volume agarose solution in the 60 °C water bath.
Prepare a 10 Mass / % volume gelatin solution: add gelatin powder to a volume of DW equal to 1.2x the volume of the 4 Mass / % volume agarose solution in a 50 mL tube.
Note
A larger volume of gelatin solution is required because some loss may occur during transfer of the viscous solution.
Place the 10 Mass / % volume gelatin solution in the 60 °C water bath for 00:20:00 to dissolve completely.
While maintaining 60 °C , mix the 10 Mass / % volume gelatin solution with the 4 Mass / % volume agarose solution at a 1:1 volume ratio using a serological pipette.
Adjust the water bath temperature to 40 °C to cool the final 2 Mass / % volume agarose–5 Mass / % volume gelatin solution.
Sample Preparation
Transfer small tissue fragments or spheroids into Microtubes and wash twice with DPBS.
Add > 20× sample volume of neutral buffered formalin (NBF) solution to the tube and fix at Room temperature for 00:10:00 -00:30:00 , depending on sample size.
Note
If the sample is not fixed before double embedding, it may be distorted by water imbibition.
Wash the sample twice with DW, each for 00:05:00 .
First Embedding
Place the sample onto a Petri dish.
Note
Transfer the samples with a small amount of water using a pipette with the tip cut off to minimize damage.
Remove excess water around the sample with tissue paper.
Note
Proceed immediately to the next step before the sample dries.
Add 200 µL of 2 Mass / % volume agarose–5 Mass / % volume gelatin solution onto the sample.
Note
Keep the area around the sample free of air bubbles.
Using pipet tips or forceps, position the sample at the center and adjust it so that the desired cutting direction faces downward.
Allow the gel to solidify at Room temperature for 00:40:00 .
Trim the solidified gel into a pyramid shape (~25 mm³ base) using a blade.
Note
The pyramid shape helps maintain the orientation of the sample during subsequent processing.
Citation
LINK
Transfer the pyramid-shaped gel into a Microtube containing 1 mL of NBF solution.
Note
Unfixed gels tend to stick together; therefore, fixation must be performed in separate Microtubes for each sample.
Fix at 4 °C for 24:00:00 .
Proceed with routine paraffin embedding according to standard procedures.
Protocol references
[1] McClelland KS, Ng ET, Bowles J. Agarose/gelatin immobilisation of tissues or embryo segments for orientated paraffin embedding and sectioning. Differentiation. 2016;91(4-5):68-71 doi:10.1016/j.diff.2015.12.001.
Citations
Step 16
McClelland KS, Ng ET, Bowles J. Agarose/gelatin immobilisation of tissues or embryo segments for orientated paraffin embedding and sectioning.
https://doi.org/10.1016/j.diff.2015.12.001