Section preparation and staining were carried out as described previously17 (link). Briefly, after the final BBB evaluation, animals were perfused with 4% paraformaldehyde solution. Spinal cord tissue (0.5 cm caudal to the injury) was harvested and placed in 4% paraformaldehyde then successively in 0.1 M phosphate buffered saline solutions that contained 10%, 20% and 30% sucrose. Spinal cord tissue was sectioned at 20 μm thickness in a freezing microtome (Leica CM1900, Germany). Every 50 sections, 1 slide was selected for immunofluorescence staining. After blocking for 1 hr with 10% normal goat serum, sections were incubated in primary antibody (eIF5A1, 1:200, Abcam; RhoGDIα, 1:200, Abcam; SMI-312(NF), 1:500, Covance; NeuN, 1:400, Abcam). Goat anti-rabbit (1:400, Invitrogen) and goat anti-mouse (1:400, Invitrogen) were used as secondary antibody. Lastly, sections were imaged with Leica AF6000 fluorescence microscope. The neurofilament positive cells (NF+) and NeuN positive cells (NeuN+) were measured by Leica LAS AF software. We used this software to compute the positive cells in spinal gray matter per mm2. The computing method of Fig. 3E,F and Fig. 6 D,E is following. Positive cells (NF+ or NeuN+) in total (%) = (positive cells number/total cells number (DAPI + nucleus)/area (mm2))*100.
Rhogdiα
Manufactured by Abcam
Sourced in United States, United Kingdom
RhoGDIα is a protein that regulates the activity of Rho GTPases, which are important signaling proteins involved in various cellular processes. RhoGDIα acts as a negative regulator of Rho GTPases by binding to them and inhibiting their interaction with downstream effectors.
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Lab products found in correlation
Las af software, by Leica (1 mentions)
Eif5a1, by Abcam (1 mentions)
Cm1900, by Leica (1 mentions)
Neuronal nuclei (neun), by Abcam (1 mentions)
Cofilin, by Proteintech (1 mentions)
Rock1, by Proteintech (1 mentions)
Cdc42, by Proteintech (1 mentions)
Gapdh, by Proteintech (1 mentions)
Enhanced chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
4 protocols using rhogdiα
1
Spinal Cord Injury Immunofluorescence Staining
2
Comprehensive Protein Analysis Protocol
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Western blot analysis was performed using standard methods. The antibodies used were as follows: RhoGDIα (1:1000; Abcam, USA), P-LIMK (1:1000; CST, USA), LIMK (1:1000; Proteintech, USA), P-cofilin (1:1000; CST, USA), cofilin (1:1000; Proteintech, USA), P53 (1:1000; CST, USA), Rac1 (1:1000; Proteintech, USA), Cdc42 (1:1000; Proteintech, USA), Pak4 (1:1000; Proteintech, USA), Rock1 (1:1000; Proteintech, USA), RhoA (1:1000; Affinity, China) and GAPDH (1:10000; Proteintech, USA). Rac1 and RhoA activity was detected by the Rac1/RhoA Pull-Down Activation Assay Biochem Kit (Cytoskeleton, USA). The Western blot bands were quantified by ImageJ software and normalized to GAPDH.
3
Tissue Microarray Analysis of RhoGDIα and TRF1
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The TMAs were constructed at the Gastrointestinal Institute of Sun Yat‐sen University. The paraffin‐embedded tissue blocks and the corresponding histological H&E‐stained slides were overlaid for tissue TMA sampling. Duplicate cylinders 1 mm in diameter were punched from representative tumor areas of individual donor tissue blocks and re‐embedded into a recipient paraffin block at a defined position using a tissue arraying instrument (MiniCore; Alphelys, Plaisir, France).
Immunohistochemistry analysis was carried out as reported. The paraffin sections were incubated with primary antibody against RhoGDIα (1:100; Abcam) or TRF1 (1:50; Abcam). For the negative control, isotype‐matched antibodies were applied. The staining intensity of each slide was separately scored for tumor by blind evaluation by two experienced pathologists using a semiquantitative IRS. The percentages of positive tumor cells were classified as follows: 1, <25% positive cells; 2, 25–50% positive cells; 3, 50–75% positive cells; and 4, >75% positive cells. The staining intensity was scored as follows: 0, negative; 1, weak; 2, moderate; and 3, strong. Multiplication of these two scores resulted in a score ranging from 0 to 12. Under these conditions, samples with IRS 0–4 and IRS 5–12 were defined as low and high expression, respectively.
Immunohistochemistry analysis was carried out as reported. The paraffin sections were incubated with primary antibody against RhoGDIα (1:100; Abcam) or TRF1 (1:50; Abcam). For the negative control, isotype‐matched antibodies were applied. The staining intensity of each slide was separately scored for tumor by blind evaluation by two experienced pathologists using a semiquantitative IRS. The percentages of positive tumor cells were classified as follows: 1, <25% positive cells; 2, 25–50% positive cells; 3, 50–75% positive cells; and 4, >75% positive cells. The staining intensity was scored as follows: 0, negative; 1, weak; 2, moderate; and 3, strong. Multiplication of these two scores resulted in a score ranging from 0 to 12. Under these conditions, samples with IRS 0–4 and IRS 5–12 were defined as low and high expression, respectively.
4
Western Blot Protein Analysis
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Total protein (40 μg) was loaded onto a 8–12% sodium dodecyl sulfate–polyacrylamide gel, and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, United States). The membranes were blocked in Tris-buffered saline +0.1% Tween-20 (TBST) containing 5% bovine serum albumin (BSA) for 1 h and then incubated with primary antibody against SDHA, SDHB, RhoGDIα, FLNA (Abcam, United Kingdom) and GAPDH (Cell Signaling Technology, United States) at 4°C overnight. GAPDH expression was used as internal control. The membranes were incubated with secondary antibody (Cell Signaling Technology, United States) for 1.5 h after three washes. The antigen–antibody complexes were detected using an enhanced chemiluminescence kit (Thermo Fisher Scientific, United States).
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