Goat secondary antibody against rabbit and mouse immunoglobulins

The antibodies used for IHC consisted of anti-CDK1 rabbit monoclonal antibody (EPR165 [ab133327]; Abcam) and anti-MAD2L1 mouse monoclonal antibody (17D10 [ab10691]; Abcam). Slides with lengths of 1~2mm formalin-fixed, paraffin-embedded tissue sections from RMS were stained manually. For IHC staining, each slide was deparaffinized and rehydrated using xylene and then washed by a graded alcohol series and phosphate buffer saline (PBS). Antigen repair was performed by boiling the tissue slice in 0.01M citric acid buffer (pH6.0) for 15min and cooling it naturally to room temperature. Endogenous peroxidase was blocked using TBS/H2O2. The sections were incubated with first antibodies against human CDK1 and MAD2L1, and then incubated with goat secondary antibody against rabbit and mouse immunoglobulins (DAKO) at 37°C for 30 min.

Serial 4-μm formalin-fixed, paraffin-embedded tissue sections from primary STS were stained manually. The antibodies used for immunohistochemical and immunofluorescence staining are listed in Supplementary Table S2.
For immunohistochemical staining, each section was dewaxed and rehydrated using xylene and then washed using a graded alcohol series. Antigen retrieval was performed for 2.5 min at high pressure using sodium citrate. The sections were incubated with primary antibodies against human CD3, CD4, CD8 or LAG-3, and then with goat secondary antibody against rabbit and mouse immunoglobulins (DAKO) for 30 min at 37°C.
For immunofluorescence staining, human tissue samples were fixed with 4% paraformaldehyde and embedded in paraffin. The sections were then dewaxed and rehydrated using xylene and washed using a graded alcohol series. Antigen retrieval was performed for 2.5 min using sodium citrate. Next, the sections were blocked with bovine serum albumin for 30 min at 37°C and incubated with LAG-3 and CD8 antibodies at 4°C overnight at the indicated dilutions. The sections were then incubated with fluorochrome-conjugated secondary antibodies (Alexa 594 anti-mouse and Alexa 488 anti-rabbit; Invitrogen).