Hi,
I have used the following protocol for HepG2 spheroid immunostaining, however the E-Cadherin staining is very weak and difficult to see.
I am using an Olympus IX70 microscope to image.
- I am unsure what changes I need to make to the protocol to improve the images?
- I am also concerned that perhaps the microscope is not powerful enough to provide good images. Also I am open to using alternative primary antibodies - perhap Beta-Catenin.
- The staining is undertaken in a wellplate at the moment. I cannot fix to slides as I am trying to adapt for microfluidics.
Any suggestions to improving images welcome
Immunostaining of HepG2 spheroids - Protocol
1. Samples fixed with 4% (v/v) paraformaldehyde (PFA) for 1 hr @ 37C, washed with PBS and then permeabilized for 1 hr with 0.3% (v/v) triton permeation solution @ RT with gentle agitation. Washed with PBS
a. Triton – take 3uL in 997uL to give you 0.3%
2. Sample blocked for 2 hrs with blocking buffer (10% BSA in PBS). Incubated overnight @ 4°C in PBS with 1.5% BSA containing primary antibodies at a suitable dilution e.g 1:500 & 1:1000.
a. 250ug/mL Anti-E-Cadherin –
a. 1uL from stock into 999uL PBS to give 0.25µg/mL
b. 10uL from stock into 990uL PBS to give 2.5µg/mL
c. 20uL from stock into 980uL PBS to give 5µg/mL
Takes 4 hrs + overnight to this step
3. The samples were washed with DPBS and incubated for 2 h with secondary antibodies @ RT in the dark at a dilution of 1:500
a. 1mg/ml Alexa-flour – 10uL of stock into 990uL in PBS to give 10ug/mL
4. The samples were then treated with DAPI (1:1000) for 15 min at 37 °C and washed briefly with DPBS before imaging.
a. DAPI 1 mg/mL – 1ul of stock into 999uL in PBS to give 1ug/mL. Prep immediately prior to use.
Takes 2 hrs 15 min + overnight to this step
Hi,
We have also done both beta-catenin and E-cad ICC in breast cancer stem cells (spheroids). Article Inhibition of breast cancer stem-like cells by a triterpenoi...
It should have worked for you. However, Some of your steps seem to have in excessive amounts, we followed generally available protocol.
*PFA: 4%; 15min; room temp. (RT)
*0.3% Triton X; 30min, RT
*5% BSA blocking; 1hr; RT
*primary Ab: 4deg; Overnight
E-cad:1:1000
Beta-catenin: 1:500
*secondary Ab: 2hr; RT; 1:500
I got papers discussing the staining of spheroids, may find something useful.
Article Quantitative three-dimensional evaluation of immunofluoresce...
Regards
Saurabh
To trouble shoot, Do you have a positive control for E-cadherin? If you do see signal in positive control, then possibly your sample is not expressing E-cad. Most of the time the problem come with permeabilization. Also, make sure to probe an other antibody to troubleshoot the antibody stability. I hope this helps.
Dear Saurabh,
Please also look at the following protocol:
Article A 3D in vitro model of differentiated HepG2 cell spheroids w...
Histological and immunohistochemical analysis. HepG2 cell spheroid suspension was collected in PBS and spun at 1,000 rpm for 5 min. The pellet was fixed in 10 % buffered formalin and embedded in paraffin. Tissue sections (3 μm) were prepared and stained with hematoxylin and eosin, PAS (Periodic acid-Schiff’s reaction) and PAS after treatment with diastase to remove glycogen. Human liver tissue was provided by Bronovo Hospital, The Netherlands, in accordance with the hospital’s code of conduct regarding use of patient-derived material. Immunohistochemical detection of cytokeratin 7 and 8 (CAM5.2, 1:20, BD Biosciences, Ref. 345779) and bile canaliculi (polyclonal CEA, 1:200, DAKO, Ref. A0115) was performed using an automated immunostainer (Benchmark ULTRA, Roche Diagnostics).
Article Characterization of a Functional C3A Liver Spheroid Model
Histological analysis
Spheroids were washed in PBS, fixed for 1 hour in 4% PFA and embedded in 2% Agarose (low EEO-Sigma Aldrich) in 4% PFA then paraffin embedded. Tissue sections were cut and stained with haematoxylin and eosin (H&E) or as previously described.39 Immunohistochemistry for Ki-67, carbamoyl-phosphate synthase 1 (CPS1) and CYP2E1 was carried out by the Department of Veterinary Pathology, Leahurst Campus, University of Liverpool, UK.
Immunofluorescence analysis of spheroids
Spheroids were transferred to ULA plates, washed three times in PBS and fixed with 4% PFA for 1 hour at 4 °C. Spheroids were washed again then permeabilized with 0.5% Triton X-100 in Tris-Buffered Saline with 0.05% Tween20 (TBST) overnight at 4 °C and then blocked with 0.1% Triton X-100/3% BSA in TBST for 2 hours at room temperature (RT). Primary antibodies Multidrug resistance protein-2 (MRP2-Abcam) and P-glycoprotein (Pgp-Abcam) were diluted 1:20 in 0.1% Triton X-100/1% BSA in TBST were incubated with the spheroids overnight at 4 °C. Spheroids underwent three 1 hour washes with 1% Triton X-100 in TBST then incubated with secondary Alexa Fluor 488 Donkey Anti-Mouse antibody (Life Technologies) diluted 11000, Hoechst diluted 1:5000 and Phalloidin 568 diluted 1:250 in 0.1% Triton X-100/1% BSA in TBST overnight at 4 °C. Spheroids were finally washed for 1 hour then mounted with Prolong Gold (Life Technologies) onto a glass microscope slide. Maximum intensity projection images of spheroids were taken using a Zeiss Axio Observer microscope with Apoptome using 40× oil objective.
Article Assembly of Multi‐Spheroid Cellular Architectures by Program...
Culture Medium
HepG2,
Immunostaining of Spheroids
The merged spheroids were fixed by paraformaldehyde (PFA, 4%) after collecting and washing with PBS (1X). Then the spheroids were permeabilized by Triton X-100 (0.5%) at room temperature for 30 min. After washing with PBS three times, the spheroids were immersed in BSA (1%) blocking solution for 2 h at room temperature. The solution was then replaced with anti-E cadherin antibody (1/500) and kept at 4 °C overnight. The excess antibodies were washed away with PBS. The Alexa Fluor 488 labeled secondary antibodies (goat anti-rabbit IgG, 1/800) was introduced at room temperature for another 1 h. The spheroids were then washed with PBS and immersed in the solution of DAPI (10 µg mL−1) for 30 min. After totally washed with PBS, the spheroids were placed in an ibidi chamber (200 µL PBS) and imaged by confocal microscopy (LSM 800, Zeiss Germany). The spheroids were settled by natural sedimentation method in each step in order to keep the original shape of the spheroids.
I am also culturing spheroids in ultra low attachment plates (ULP).
I cultured them successfully but the concentration of RNA came out to be low. May I know after culturing in ULP, i need to transfer the fully grown spheroids after trypsinization into normal plates and then isolate RNA from them. Alexandr Chernov Can you please elaborate?
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